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We investigated the involvement of inhibitory innervation in reflex tracheal dilatation (RTD) induced by inflating the lungs in dogs. RTD was inhibited about 50% by 100 micrograms propranolol injected into the cranial thyroid artery, but was unaffected by adrenalectomy. Residual RTD under beta-blockade was abolished by sections of both the bilateral superior laryngeal nerves and spinal cord at the C1 level. These findings suggest that RTD may be mediated by adrenergic innervation and partly by nonadrenergic inhibitory innervation.

We have designed a new method for continuous recording of the output of respiratory tract fluid, the approach representing a modification and combination of the intratracheal electrode and stopper methods which we had employed originally. Electrical resistance change indicated alteration in the output of respiratory tract fluid, without alteration in composition and temperature of fluid, tracheal muscular tone or respiratory movement, at least, within a physiological range. Among the expectorant drugs thus tested, pilocarpine, 100 and 200 micrigrams/kg given intravenously, significantly and in a dose dependent manner augmented the output of the fluid about 5 minutes after the administration. Given orally in a dose of 2 mg/kg, the output of fluid increased in about 20 minutes. Senega syrup, 0.3 ml/kg had no effect when given intravenously, yet, 2 ml/kg given orally increased the output of fluid within 5 minutes, thereby suggesting that the related secretagogic activity is due to a reflex action following stimulation of the gastric mucosa. Emetine, 2 mg/kg given orally increased the output about 20 minutes after administration while glyceryl guaiacolate, 50 mg/kg had no effect. We propose that our method be used to evaluate expectorants for clinical use as the continuous monitoring of the output of respiratory tract fluid apparently provides a more accurate assessment.

The mechanism for the expectorant effect of S-carboxymethylcysteine (S-CMC) was investigated histologically and histochemically using isolated canine trachea. Following S-CMC treatment, the number of total glycoprotein-containing goblet cells (GC) did not change. The numbers of acid glycoprotein (AGP)-, neutral glycoprotein (NGP)-, and sulphated glycoprotein (SGP)-containing GC were also unaltered in a concentration range of 10(-7) to 10(-4) M. On the other hand, the ratio of the acinar inner diameter of submucosal glands (SG) to the tracheal wall thickness significantly increased with 10(-5) and 10(-4) M S-CMC, and the thickness of the acini of SG significantly decreased with 10(-4) M S-CMC. Although the ratio of the numbers of AGP-to NGP-containing glandular cells and AGP content in the cells did not change, the number of SGP-containing glandular cells significantly decreased with 10(-4) M S-CMC, and SGP content in the cells significantly decreased concentration-dependently High concentrations (10(-5) and 10(-4) M) of S-CMC produced increases in total saccharide, protein and N-acetylhexosamine concentrations in the incubation fluid. These findings suggest that S-CMC has a selective secretagogic action on SG, and an action which alters the composition of AGP, a chief viscous factor, in the mucous granules of SG. The expectorant effect of this agent may, at least in part, be ascribable to these actions.

The effects of glyceryl guaiacolate on secretory activities of tracheal secretory cells and on behavior of mucus glycoprotein in these cells were investigated histologically and histochemically using isolated canine trachea. Following glyceryl guaiacolate treatment, the number of total glycoprotein-containing goblet cells (GC) did not change. The numbers of acid glycoprotein (AGP)-, neutral glycoprotein (NGP)-, and sulphated glycoprotein (SGP)-containing GC were also unaltered in a concentration range of 10(-7) to 10(-4)M. On the other hand the acinar inner diameter of the submucosal gland (SG) and the acinar inner diameter to wall ratio significantly increased, while thickness of acinus significantly decreased with 10(-6), 10(-5) and 10(-4)M glyceryl guaiacolate. AGP and SGP contents in glandular cells markedly decreased, while the ratio of the numbers of AGP- to NGP-containing glandular cell were the same, suggesting that glyceryl guaiacolate does not change the quality of mucus glycoprotein in GC and SG. Glyceryl guaiacolate produced a marked increase in total saccharide, protein, and N-acetylhexosamine concentrations in the incubation fluid. These findings suggest that glyceryl guaiacolate has no influence on the secretory activity of GC, but markedly stimulates the activity of SG. The secretagogue effect of the agent would be ascribable to stimulation of mucus discharge, but not stimulation of mucus synthesis.

Behavior of mucus glycoproteins in tracheal secretory cells after treatment with pilocarpine was investigated histologically and histochemically using the isolated canine trachea. Following pilocarpine treatment, the number of total and neutral glycoprotein-containing goblet cells was reduced concentration-dependently. The number of acid glycoprotein (AGP)-containing goblet cells was not altered with 10(-7) and 10(-6) M pilocarpine, but significantly decreased with 10(-5) and 10(-4) M pilocarpine. The thickness of the acini of submucosal glands significantly decreased, and the ratio of acinar inner diameter to tracheal wall thickness increased in 10(-5) and 10(-4) M pilocarpine. AGP content in glandular cells increased in 10(-7) and 10(-6) M pilocarpine, but markedly decreased at concentrations of 10(-5) and 10(-4) M. Pilocarpine treatment caused an increase in N-acetylhexosamine concentration in the incubation fluid. Total saccharide concentration in the incubation fluid decreased in 10(-7) and 10(-6) M pilocarpine, but was not apparently altered at concentrations of 10(-5) and 10(-4) M. These findings suggest that lower concentrations of pilocarpine stimulate synthesis of AGP in goblet and glandular cells much more preferentially than it stimulates discharge of AGP from the cells. While at higher concentrations, the AGP-discharge effect overcomes the stimulation in synthesis.

The effects of brovanexine (BvX) and BR-227 on sectetory activities of tracheal submucosal glands (SG) including behavior of mucus glycoproteins in the cells were investigated. BvX, BR-227 or bromhexine (BH) was given repeatedly at a dose of 10 or 20 mg/kg p.o. a day, to rats for 1, 3, 7 or 14 days. Then, the trachea was excised and drug effects were determined according to our histological/histochemical technique. The outer diameter of the acini of SG did not change, but the inner diameter markedly increased by the drug given for 3-14 days. The ratio of the acinar inner diameter to the tracheal wall thickness (AIWR) was increased with the 3, 7, and 14 day treatments with BvX or BR-227 at a dose of 10 mg/kg a day. On the other hand, these drugs at a dose of 20 mg/kg a day caused an increase in AIWR when drug was given for 1-14 days. BH (10 mg/kg a day)-induced increase in AIWR was shown when drug was administered for 7 and 14 days. The number of SG cells stained blue with alcian blue (pH 2.5)/periodic acid-Schiff decreased with the 1, 3, 7 and 14 day treatments, and a part of SG cells became red with the 7- and 14-day treatments. There was no significant difference in the effects among the three drugs both qualitatively and quantitatively. These findings indicate that repeated treatments with BvX and BR-227 have an effective secretagogic action on SG, and in addition, a mucolytic action toward acid glycoprotein in granules of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

We devised a canine blood-perfusion preparation which made feasible administration of drugs directly at the local tracheal site. The hypothesis of Salem and Aviado on cough mechanism that a local airway constriction induced by stimuli may be a trigger in stimulating cough receptors was investigated using this preparation. Close intraarterial injections of acetylcholine (ACh) and histamine did not elicit a cough although intense tracheal constrictions were evident. The cough reflex elicited by electrical stimulation of the mucosa of isolated upper trachea in situ was accompanied by a slight systemic hypotension, tracheal vasodilatation and tracheal muscular constriction. The latter two changes occurred after a time lag following coughs. Close intraarterial infusions of isoproterenol and papaverine caused a prominent tracheal dilatation, but did not suppress the coughs. Pretreatment with atropine sufficiently inhibited cholinergic tracheoconstriction but had no effect on the electrically induced coughs. Furthermore, an augmentation of the tracheal muscular tone produced by an infusion of ACh did not enhance the cough reflex. In light of our observations, the aforementioned hypothesis should be reconsidered.

We investigated the effect of beta-adrenoceptor stimulants on the canine tracheal ciliated cells. A mucosal specimen of the tracheal membranous cell was placed in a chamber containing Hanks' solution. Membrane potential was recorded by an intracellular microelectrode, and ciliary beating was measured by a photoelectrical technique. Isoproterenol and salbutamol in 10(-7) to 10(-4) M caused a concentration-dependent increase in both frequencies of ciliary beating and small electrical oscillatories were superimposed on the membrane potential. Theophylline also increased frequencies of both activities in doses of 10(-5) M and above. Onset of action of theophylline was later than that of isoproterenol or salbutamol. All the above drugs only slightly depolarized the membrane potential, in concentrations of 10(-4) M. Propranolol (10(-7)-10(-4) M) alone had little effect on ciliary beating and electrical activity. However, the effects of isoproterenol and salbutamol were effectively antagonized by propranolol (10(-5) M). Dibutyryl cyclic AMP (10(-7)-10(-4) M) caused concentration-dependent increases in ciliary beating and electrical activity. These results suggest that potent bronchodilators such as beta-stimulants and theophylline increase the activity of tracheal ciliated cells by stimulating the intracellular cyclic AMP system.

The effects of BR-227 on secretory activities of canine tracheal secretory cells including behavior of mucus glycoproteins were investigated histologically and histochemically. Following BR-227 treatment at a concentration of alcian blue at pH 2.5 and periodic acid-Schiff (AB (pH 2.5)/PAS) decreased in a concentration-dependent manner. Furthermore, a decrease in the thickness of the acini of submucosal glands and in marked increase in the ratio of acinar inner diameter of the gland to tracheal wall were induced after application of BR-227 at a concentration range of 10(-7) to 10(-4) M. The numbers of goblet and submucosal glandular cells which stained blue and purple with AB (pH 2.5)/PAS were decreased by BR-227 treatment in a concentrations-dependent way, whereas the cells which stained red were markedly increased. In the experiment using a combination of AB at pH 1.0 and PAS, a decrease in sulfated glycoproteins in those secretory cells was observed. Total saccharide and protein concentrations in the incubation fluid increased with BR-227 treatment, while N-acetylhexomsamine concentration tended to decrease. These findings suggest that BR-227 stimulates secretory activities of both goblet cells and submucosal glands, and lowers mucus viscosity in the secretory cells.

The expectorant effect of brovanexine (BvX) was investigated histologically and histochemically using isolated canine trachea. Following BvX treatment, the number of goblet cells (GC) stained positively with the combination procedure of alcian blue at pH 2.5 or pH 1.0 and periodic acid-Schiff was unaffected. The number of GC which stained blue and purple (stain index B & P) was reduced, while the number of GC that stained red (stain index R) was increased. The thickness of the acini of submucosal glands (SG) markedly decreased with 10(-5) and 10(-4)M BvX, and the ratio of the acinar inner diameter to the tracheal wall thickness significantly increased with 10(-4)M BvX. Stain index B & P of glandular cells decreased with 10(-5) and 10(-4)M BvX, which was accompanied by a marked increase in the stain index R. BvX treatment caused increases in total saccharide and protein concentrations in the incubation fluid, while N-acetylhexosamine slightly decreased. Both the BvX-induced histological changes in glandular cells and changes in concentrations of macromolecular components in the incubation fluid were much the same degree as those induced by bromhexine (Bh). The BvX-induced histochemical changes in secretory cells, however, were slighter than those induced by Bh. These findings suggest that BvX is an expectorant possessing both the secretagogic action selectively on SG and a mucolytic action toward acid glycoproteins in granules of secretory cells.

The effects of L-cysteine methyl ester on behavior of mucus glycoproteins in canine tracheal secretory cells were investigated histologically and histochemically. Low concentrations (10(-7) and 10(-6)M) of L-cysteine methyl ester produced, as histological changes, decreases in the total goblet cell number and the thickness of the acini of submucosal glands, and an increase in the acinar inner diameter. On the other hand, L-cysteine methyl ester at 10(-5) and 10(-4)M produced an increase in the thickness of the acini and a slight decrease in the acinar inner diameter. Both the total acid glycoprotein and sulphated glycoprotein contents in glandular cells markedly decreased, whereas the neutral glycoprotein content in the cells increased following L-cysteine methyl ester treatment in a concentration-dependent way. Total saccharide and protein concentrations in the incubation fluid increased with 10(-7) and 10(-6)M, but decreased with 10(-5) and 10(-4)M L-cysteine methyl ester. N-Acetylhexosamine concentration in the bathing fluid increased at 10(-7) and 10(-6)M of the agent. These findings suggest that low concentrations of L-cysteine methyl ester stimulate secretory activities of tracheal secretory cells, while at high concentrations the agent stimulates synthesis of mucus glycoproteins in the submucosal glands. Additionally, it is suggested that L-cysteine methyl ester has a marked viscosity lowering effect on the mucus in the secretory cells.

Many expectorants are clinically effective as they reduce viscosity and facilitate expectoration. There are, however, few reports on effects of expectorants on tracheal ciliated cells. We investigated the effects of N-acetylcysteine, ethylcysteine and bromhexine on the ciliary activities of the canine trachea. Ciliary movement was estimated using a phototransistor, and intracellular electrical activity was measured with a microelectrode method, in vitro. N-Acetylcysteine, ethylcysteine and bromhexine in low concentrations under a low perfusion rate (0.1 ml/min) produced an increase in the amplitude and frequency of ciliary beating, while N-acetylcysteine and ethylcysteine caused a cilio-depression in high concentration. N-Acetylcysteine, ethylcysteine and bromhexine, under a low perfusion rate, did not affect the intracellular electrical activity. On the other hand, these three drugs under a high perfusion rate (1 ml/min) produced no change in the ciliary movement and the intracellular electrical activity in concentrations of 10(-8)-10(-4) M. These results suggest that the increase in ciliary activity produced by the mucolytic drugs is not due to a direct effect on the ciliated cells, but rather to a mucolytic effect on the mucus around the cilia.

The effects of bromhexine on secretory activities of canine tracheal secretory cells with stimulated synthesis of acid glycoprotein (AGP) by pilocarpine 10(-6)M treatment an on behaviour of glycoprotein in these secretory cells were investigated histologically and histochemically. Following bromhexine treatment, the number of total glycoprotein-containing goblet cells (GC) remained unchanged. The numbers of AGP-containing and sulphated glycoprotein (SGP)-containing GC significantly decreased, while neutral glycoprotein (NGP)-containing GC significantly increased. The acinar inner diameter of the submucosal gland and the acinar inner diameter of this gland to wall ratio slightly increased. Thickness of the acinus and Reid index slightly decreased, concentration-dependently. AGP and SGP content in glandular cells decreased, while NGP content in these cells markedly increased. Bromhexine 10(-5) and 10(-4)M treatment resulted in increased total saccharide and protein concentrations in the incubation fluid, whereas the agent significantly decreased N-acetylhexosamine, in a concentration-dependent manner. These findings suggest that while bromhexine does not stimulate secretory activities of GC, it does slightly stimulate the activities of the submucosal glands. Bromhexine markedly dissolves AGP in the granules of these secretory cells.

The effects of bromhexine, a mucolytic agent, on secretory activities of canine tracheal secretory cells and on behaviour of glycoprotein in these secretory cells were investigated histologically and histochemically. Following bromhexine treatment, the number of total glycoprotein-containing goblet cells (GC) remained the same. The number of acid glycoprotein (AGP)-containing GC was reduced concentration-dependently, while neutral glycoprotein (NGP)-containing GC significantly increased with doses of 10-6, 10-5 and 10-4M. The number of sulphated glycoprotein (SGP) in AGP-containing GC decreased concentration-dependently. Acinar inner diameter of the submucosal gland and an acinar inner diameter of this gland to wall ratio (AIWR) increased with doses of 10-5 and 10-4M. Particularly, AIWR significantly increased with dose of 10-4M. Thickness of the acinus tended to slightly decrease concentration-dependently. AGP content in glandular cells decreased, while NGP content in these cells markedly increased, concentration-dependently. SGP content in the glandular cells decreased. Total saccharide and protein concentrations in the incubation fluid significantly increased with higher concentrations of the treatment, whereas N-acetylhexosamine slightly decreased concentration-dependently. These findings suggest that bromhexine does not enhance secretory activities of GC, but does stimulate the activities of the submucosal glands, when the drug is given in higher concentrations. Bromhexine markedly dissolves AGP in granules of the secretory cells.

The effect of bromhexine on behaviors of lysosomal enzymes in the submucosal gland was investigated using canine tracheal slice preparations, with reference to the histochemical changes in acid glycoproteins (AGP) in the gland. Incubation of tracheal slices with 1% Triton X-100 or 0.0004-0.04% bromhexine for 30 min decreased the number of stained lysosomes in the glandular cells. The decrease in stained lysosomes after treatment with 1% Triton X-100 or 0.04% bromhexine was effectively prevented by addition of 5% lecithin. The number of glandular cells that were stained red (stain index R) in the combined alcian blue at pH 2.5 and periodic acid-Schiff procedure markedly increased by treatment with 1% Triton X-100 or 0.0004-0.04% bromhexine. In the bromhexine treated groups, there was a close correlation (r=0.932, P less than 0.01) between the increase in the number of glandular cells showing the stain index R and the decrease in the number of stained lysosomes in the cells. These findings suggest that the enzymes which are liberated from lysosomes into the cytoplasm by bromhexine may, at least in a part, be involved in the mucolytic action of the agent on AGP contained in mucus granules of the submucosal gland.

In a previous paper, it was shown that the cough reflex was accompanied by a slight fall of systemic arterial pressure, tracheal constriction and tracheal vasodilation. In the present study, tracheobronchial muscular and vascular tones during the cough reflex were investigated using the blood perfused canine tracheal and bronchial preparations in situ. The cough reflex was elicited with electrical stimulation of the membraneous wall mucosa of the upper trachea. In the blood perfused tracheal preparation, a close intraarterial injection of atropine or bilateral vagotomy inhibited the tracheoconstriction but had no effect on the tracheal vasodilation during the cough reflex. In the blood perfused bronchial preparation, electrical stimulation of the upper tracheal mucosa induced bronchial constriction but not bronchial vasodilation. Lung inflation with air resulted in a bronchial vasodilation and which persisted even after a close intraarterial infusion of atropine and benzonatate and after bilateral vagotomy. These findings indicate that the tracheobronchial constriction response are primary reflex effects following coughing and the tracheobronchial vasodilation responses are secondary reflex effects induced by increase in internal pressure of the respiratory tract.

We devised a preparation for measuring the vagal reflex tracheal constriction following the bronchoconstriction induced by histamine inhaled in the bronchial side in dogs. Properties of the vagal reflex tracheal constriction were investigated using this preparation. Histamine inhaled in the bronchial side caused the tracheal constriction following the bronchoconstriction. The tracheal constriction was inhibited by section of the bilateral superior laryngeal nerves or vagal cooling, respectively, but was not completely blocked. The combination of section of the bilateral superior laryngeal nerves and vagal cooling abolished the tracheal constriction. An i.v. administration of pentobarbital reduced both bronchoconstriction and tracheal constriction. These findings indicate that the tracheal constriction observed in the present study is mediated by the vagal reflex arc and that the extravagal pathway consisting of the recurrent and superior laryngeal nerves plays a role in a part of the afferent pathway of the vagal reflex airway responses. When the bronchoconstriction was completely abolished by isoproterenol inhaled in the bronchial side, the reflex tracheal constriction still existed. Transient inflation and deflation of the lungs caused reflex tracheal dilatation and constriction, respectively. We conclude that the vagal reflex airway constriction is due to complex effects which may be mediated by plural sensory receptors in the airways.

The effects of brovanexine (BvX) on secretory activities of tracheal secretory cells and on behavior of mucus glycoprotein in these cells were investigated histologically and histochemically using the biopsy technique. When BvX was given at 10 or 20 mg/kg intraduodenally to anesthetized dogs, the thickness of the acini of submucosal glands (SG) and the ratio of the acinar inner diameter to the tracheal wall thickness (A1WR) markedly increased dose-dependently after 2 to 6 hr. The numbers of goblet cells (GC) and glandular cells showing a stain index B&P with a combination of alcian blue (AB) at pH 2.5 or pH 1.0 and periodic acid-Schiff (PAS) were reduced dose-dependently after 1 to 6 hr, while the number of cells with a stain index R were increased. Both the BvX-induced histological changes in SG and histochemical changes in GC and SG reached a peak after 4 hr. These histological and histochemical changes were also found for the BR-227-, a metabolite of BvX, and bromhexine (BH)-treated groups. BR-227- or BH-induced histological changes were to the same degree as those induced by BvX, but BvX- or BR-227-induced histochemical changes were slighter than those induced by BH. The total number of GC stained positively with a combination of AB at pH 2.5 and PAS was unaffected by BvX or BH treatment, while BR-227 tended to decrease it. These findings suggest that BvX both has a secretagogic action selectively on SG and a mucolytic action toward acid glycoprotein in granules of secretory cells in vivo, and these actions are durable.