Cigarette smoke-induced bronchoconstriction in dogs: vagal and extravagal mechanisms

We reassessed the severity of cigarette smoke-induced bronchoconstriction and the mechanisms involved in anesthetized dogs. To evaluate the severity of smoke-induced bronchoconstriction, we measured airway pressure and airflow resistance (Rrs, forced oscillation method). We studied the mechanisms in other dogs by measuring airway pressure, central airway smooth muscle tone in tracheal segments in situ, and respiratory center drive by monitoring phrenic motor nerve output, including the role of vagal and extravagal nerves vs. the role of blood-borne materials during inhalation of cigarette smoke. Rrs increased more than fourfold with smoke from one cigarette delivered in two tidal volumes. About half the airway response was due to local effects of smoke in the lungs. The remainder was due to stimulation of the respiratory center, which activated vagal motor efferents to the airway smooth muscle. Of this central stimulation, about half was due to blood-borne materials and the rest to vagal pulmonary afferents from the lungs. We conclude that inhalation of cigarette smoke in dogs causes severe bronchoconstriction which is mediated mainly by extravagal mechanisms.     

Influence of nicotine in cigarette smoke on acute ventilatory responses in awake dogs

To determine whether the acute ventilatory responses to inhaled cigarette smoke are affected by a difference in nicotine level, control cigarettes (low-nicotine research cigarettes) were laced with nicotine to generate an increase of 330% (mean) in nicotine content with little or no change in the levels of other smoke constituents. Acute ventilatory responses to both control and nicotine-laced cigarettes were determined and compared in six awake chronic dogs. Spontaneous inhalation of nicotine-laced cigarette smoke (10% concn, 750 ml vol) via a tracheostomy tube caused distinct and consistent changes in breathing pattern on the first or second breath of inhaled smoke: an apnea in three dogs, an augmented inspiration in two dogs, and rapid shallow breathing in one dog. No significant change in breathing pattern was found immediately following inhalation of control cigarette smoke. Both types of cigarettes caused a delayed hyperpnea. However, the increase in minute ventilation induced by nicotine-laced cigarettes (from a base line of 2.8 to a peak of 25.7 l/min) was significantly greater than that by control cigarettes (from 2.9 to 5.5 l/min). Results of this study suggest that nicotine is responsible for the elicitation of both the immediate and delayed ventilatory responses to inhaled cigarette smoke generated under our experimental conditions.     

Chest wall distortion and discharge of pulmonary slowly adapting receptors.

We assessed the effects of chest wall distortion, changes in lung volume, and abolition of airway smooth muscle tone on the discharge patterns of 92 pulmonary slowly adapting receptors (SAR) in decerebrate, spontaneously breathing cats. Distortion resulted from their inspiratory efforts against an occluded airway at functional residual capacity and at increased end-expiratory lung volumes. Approximately 40% of SAR increased discharge frequencies during occlusions. Modulation of SAR discharge during occlusions persisted after administration of atropine to eliminate airway smooth muscle tone. Phasic modulation of SAR discharge was eliminated during no-inflation tests after paralyzing the cats and ventilating them on a cycle-triggered pump. We conclude 1) parasympathetic modulation of airway smooth muscle tone makes no obvious contribution to SAR discharge in spontaneously breathing cats; 2) the no-inflation test (withholding of lung inflation during neural inspiration) in paralyzed and ventilated cats is a valid test for the presence of projections from SAR to medullary respiratory neurons; and 3) in the absence of tidal volume changes, distortion stimulates some SAR. Sensory feedback from receptors in the lung, not just those in the chest wall, may therefore provide information about abnormal chest wall configurations.     

Cigarette smoke in lungs evokes reflex increase in tracheal submucosal gland secretion in dogs.

Stimulation of pulmonary C-fibers (PCs) by capsaicin and of rapidly adapting receptors (RARs) by reduced lung compliance reflexly increases airway submucosal gland secretion in dogs. Because both PCs and RARs are stimulated by cigarette smoke (nicotine being the primary stimulus), we performed experiments in anesthetized open-chest artificially ventilated dogs (with aortic nerves cut) to determine whether cigarette smoke reflexly stimulates airway secretion. We measured submucosal gland secretion by counting the hillocks in a 1.2-cm2 field of tracheal epithelium coated with tantalum dust. Secretion was stimulated by delivery of 40-320 ml smoke from high-nicotine cigarettes to the lower trachea, secretion rate increasing from 7.4 +/- 1.3 to 48.1 +/- 5.1 hillocks.cm-2.min-1. Results of cutting the pulmonary vagal branches or carotid sinus nerves or both indicated that the secretory response was initiated by stimulation of lower respiratory vagal afferents and augmented several seconds later by stimulation of carotid chemoreceptors. Results of cooling the cervical vagus nerves to 7 and 0 degrees C indicated that most of the vagally mediated increase in secretion was due to stimulation of afferent lung C-fibers.     

Acute effects of cigarette smoke inhalation on peripheral airways in dogs

We studied the acute effects of the inhalation of cigarette smoke on the central and peripheral airways of 35 open-chested and tracheotomized dogs by the direct measurement of central (Rc) and peripheral (Rp) airway resistances. Rc was calculated by dividing the pressure difference between a tracheal catheter and a retrograde catheter by mouth flow, and Rp was obtained by dividing the pressure difference between the retrograde catheter and a pleural capsule by mouth flow. The pleural capsule was attached to the pleural surface for alveolar pressure measurement. Rc and Rp were measured by the 2-Hz forced oscillation method. With lung inhalation of the smoke of two-thirds of one cigarette in vagi intact dogs, Rp increased to 239% of the control value and Rc increased to 112%. After bilateral vagotomy, Rp increased to 143% and Rc increased to 104%. Propranolol did not influence the results. Hexamethonium and atropine both blocked these responses when vagi were intact. When the upper trachea, larynx, and nasopharynx, which were completely blocked by vagotomy, were exposed to the smoke of two-thirds of a cigarette, Rp increased to 155% and Rc increased to 144%. We thus conclude that cigarette smoke causes a major increase in Rp, mainly via the vagal reflex and partially via the stimulation of parasympathetic ganglia (probably nicotine), and a minor increase in Rc via vagal reflex.     

Stimulation of vagal pulmonary C-fibers by a single breath of cigarette smoke in dogs

Inhalation of cigarette smoke into the lower airway via a tracheostomy evokes immediate apnea, bradycardia, and systemic hypotension in dogs. These responses can still be evoked when conduction in myelinated vagal fibers is blocked preferentially by cooling but are abolished by vagotomy, suggesting that they are mediated by afferent vagal C-fibers. To examine this possibility, we recorded impulses in pulmonary C-fibers in anesthetized, open-chest dogs and delivered 120 ml cigarette smoke to the lungs in a single ventilatory cycle. Pulmonary C-fibers were stimulated within 1 or 2 s of the delivery of smoke generated by high-nicotine cigarettes, activity increasing from 0.3 +/- 0.1 to a peak of 12.6 +/- 1.3 (SE) impulses/s, (n = 60); the evoked discharge usually lasted 3-5 s. Smoke generated by low-nicotine cigarettes evoked a milder stimulation in 33% of pulmonary C-fibers but did not significantly affect the overall firing frequency (peak activity = 2.2 +/- 1.1 impulses/s, n = 36). Hexamethonium (0.7-1.2 mg/kg iv) prevented C-fiber stimulation by high-nicotine cigarette smoke (n = 12) but not stimulation by right atrial injection of capsaicin. We conclude that pulmonary C-fibers are stimulated by a single breath of cigarette smoke and that nicotine is the constituent responsible.     

Tracheobronchial muscle tonus and its reflex control

Airway smooth muscle tone is reinforced during the inspiratory phase of the breathing cycle and depends largely from neurogenic motor drive carried by the vagus nerve. This muscle tone seems to be produced mostly by a vago-vagal reflex loop initiated by the tonic discharge of tracheo-bronchial and/or alveolar receptors connected to thin sensory vagal fibres (non-myelinated or C-fibres). Inhibitory influences carried by large myelinated vagal fibres connected to tracheobronchial stretch receptors and also numerous afferents from the upper airways, systemic and pulmonary circulation, digestive tract and skeletal and respiratory muscles participate to the modulation of airway tone. The identification of neurotransmitters specific of the motor or sensory pathways helps to understand the peripheral modulation of airway motor drive and also the central integration of some peripheral informations.     

Acute effects of cigarette smoke on breathing in rats: vagal and nonvagal mechanisms

The acute ventilatory response to inhalation of cigarette smoke was studied in anesthetized Sprague-Dawley rats. Cigarette smoke (6 ml, 50%) generated by a machine was inhaled spontaneously via a tracheal cannula. Within the first two breaths of smoke inhalation, a slowing of respiration resulting from a prolonged expiratory duration (173 +/- 6% of the base line; n = 32) was elicited in 88% of the rats studied. This initial inhibitory effect on breathing was not affected either by an increase (410%) in the nicotine content of the cigarette smoke or by pretreatment with hexamethonium (33 mg/kg iv). However, bilateral vagotomy completely eliminated the initial ventilatory inhibition. Cooling both vagi to 5.1 degrees C blocked the reflex apneic response to lung inflation, but it did not abolish the inhibitory effect of smoke. After the initial response, a rapid shallow breathing pattern developed and reached its peak 5-12 breaths after inhalation of high-nicotine cigarette smoke; this delayed response could not be prevented by vagotomy and was undetectable after inhalation of low-nicotine smoke. We conclude that the initial inhibitory effect of smoke on breathing is mediated by vagal bronchopulmonary C-fiber afferents, which are stimulated by smoke constituents other than nicotine, whereas the delayed tachypneic response to smoke is caused by the absorbed nicotine.     

Relationship between cigarette yields,puffing patterns,and smoke intake: evidence for tar compensation?

The relationship between cigarette yields (of nicotine, tar, and carbon monoxide), puffing patterns, and smoke intake was studied by determining puffing patterns and measuring blood concentrations of nicotine and carboxy-haemoglobin (COHb) in a sample of 55 smokers smoking their usual brand of cigarette. Regression analyses showed that the total volume of smoke puffed from a cigarette was a more important determinant of peak blood nicotine concentration than the nicotine or tar yield of the cigarette, its length, or the reported number of cigarettes smoked on the test day. There was evidence of compensation for a lower tar yield over and above any compensation for nicotine. When nicotine yield was controlled for, smokers of lower-tar cigarettes not only puffed more smoke from their cigarettes than smokers of higher-tar cigarettes but they also had higher plasma nicotine concentrations, suggesting that they were compensating for the reduced delivery of tar by puffing and inhaling a greater volume of smoke. The results based on the COHb concentrations were consistent with this interpretation. If an adequate intake of tar proves to be one of the main motives for smoking, then developing a cigarette that is acceptable to smokers and also less harmful to their health will be much more difficult.     

Menthol Attenuates Respiratory Irritation and Elevates Blood Cotinine in Cigarette Smoke Exposed Mice

Addition of menthol to cigarettes may be associated with increased initiation of smoking. The potential mechanisms underlying this association are not known. Menthol, likely due to its effects on cold-sensing peripheral sensory neurons, is known to inhibit the sensation of irritation elicited by respiratory irritants. However, it remains unclear whether menthol modulates cigarette smoke irritancy and nicotine absorption during initial exposures to cigarettes, thereby facilitating smoking initiation. Using plethysmography in a C57Bl/6J mouse model, we examined the effects of L-menthol, the menthol isomer added to cigarettes, on the respiratory sensory irritation response to primary smoke irritants (acrolein and cyclohexanone) and smoke of Kentucky reference 2R4 cigarettes. We also studied L-menthol’s effect on blood levels of the nicotine metabolite, cotinine, immediately after exposure to cigarette smoke. L-menthol suppressed the irritation response to acrolein with an apparent IC₅₀ of 4 ppm. Suppression was observed even at acrolein levels well above those necessary to produce a maximal response. Cigarette smoke, at exposure levels of 10 mg/m³ or higher, caused an immediate and marked sensory irritation response in mice. This response was significantly suppressed by L-menthol even at smoke concentrations as high as 300 mg/m³. Counterirritation by L-menthol was abolished by treatment with a selective inhibitor of Transient Receptor Potential Melastatin 8 (TRPM8), the neuronal cold/menthol receptor. Inclusion of menthol in the cigarette smoke resulted in roughly a 1.5-fold increase in plasma cotinine levels over those observed in mice exposed to smoke without added menthol. These findings document that, L-menthol, through TRPM8, is a strong suppressor of respiratory irritation responses, even during highly noxious exposures to cigarette smoke or smoke irritants, and increases blood cotinine. Therefore, L-menthol, as a cigarette additive, may promote smoking initiation and nicotine addiction.     

Acute effect of cigarette smoke on laryngeal receptors

Upper airway exposure to cigarette smoke elicits reflex changes in breathing pattern. To examine whether laryngeal afferents are affected by cigarette smoke, neural activity was recorded from the peripheral cut end of superior laryngeal nerve in anesthetized dogs. A box-balloon system, connected to the breathing circuit, allowed smoke to be inhaled spontaneously through the isolated upper airway while preserving its normal respiratory flow and pressure. Our results showed the following. Inhalation of cigarette smoke (25-50% concentration, 300-400 ml) caused a marked increase in activity of laryngeal irritant receptors which were either silent or randomly discharging during control breathing [their activity increased from a control value of 1.67 +/- 0.50 (mean +/- SE; n = 21) to a peak of 5.03 +/- 0.85 impulses/s in 11-15 s]. The activity of laryngeal cold receptors was reduced to 77.3 and 63.8% of control (n = 9) during the two breaths of smoke inhalation, respectively. After returning toward the base-line activity, a more pronounced inhibition (26.3% of control) occurred at three to nine breaths after the smoke inhalation. A small but significant decrease (88.5% of control) in the inspiratory discharge of laryngeal mechanoreceptors was observed during the first test breath. These effects were independent of the CO2 content of the smoke. Furthermore, there was no difference between the responses of these laryngeal afferents to high- and low-nicotine cigarette smoke.     

Absence of nonadrenergic noncholinergic relaxation in the cat cervical trachea

Published in vivo experiments have not supported in vitro reports of the presence of nonadrenergic noncholinergic (NANC) inhibitory pathways in the cat trachea. We therefore examined these pathways, measuring tension in an innervated tracheal segment, flow resistance in more distal airways, and dynamic compliance, in 10 anesthetized mechanically ventilated cats. Initially, cervical vagal stimulation evoked contraction followed by relaxation of smooth muscle of trachea and lower airways; sympathetic stimulation evoked relaxation only. After muscarinic blockade and restoration of smooth muscle tone with 5-hydroxytryptamine (5-HT) applied topically to the tracheal mucosa, vagal stimulation did not affect tracheal segment tension, whereas sympathetic-evoked relaxation was preserved. Similar results were found when tone was restored with intravenous 5-HT, with vagal stimulation also decreasing resistance and increasing compliance. We conclude that NANC pathways are present in lower airways but not in the cervical trachea of the cat. We hypothesize that parasympathetic constriction of cat airway smooth muscle can occur without simultaneous NANC activation, whereas NANC activity occurs only in tandem with parasympathetic stimulation.     

Reflex regulation of airway smooth muscle tone.

Autonomic nerves in most mammalian species mediate both contractions and relaxations of airway smooth muscle. Cholinergic-parasympathetic nerves mediate contractions, whereas adrenergic-sympathetic and/or noncholinergic parasympathetic nerves mediate relaxations. Sympathetic-adrenergic innervation of human airway smooth muscle is sparse or nonexistent based on histological analyses and plays little or no role in regulating airway caliber. Rather, in humans and in many other species, postganglionic noncholinergic parasympathetic nerves provide the only relaxant innervation of airway smooth muscle. These noncholinergic nerves are anatomically and physiologically distinct from the postganglionic cholinergic parasympathetic nerves and differentially regulated by reflexes. Although bronchopulmonary vagal afferent nerves provide the primary afferent input regulating airway autonomic nerve activity, extrapulmonary afferent nerves, both vagal and nonvagal, can also reflexively regulate autonomic tone in airway smooth muscle. Reflexes result in either an enhanced activity in one or more of the autonomic efferent pathways, or a withdrawal of baseline cholinergic tone. These parallel excitatory and inhibitory afferent and efferent pathways add complexity to autonomic control of airway caliber. Dysfunction or dysregulation of these afferent and efferent nerves likely contributes to the pathogenesis of obstructive airways diseases and may account for the pulmonary symptoms associated with extrapulmonary disorders, including gastroesophageal reflux disease, cardiovascular disease, and rhinosinusitis.     

On the Influence of Moisture Content in Cigarettes on Combustion Temperature and Transferred Amount of Nicotine into Cigarettes Smoke

The effects of moisture content of cigarettes on both combustion temperature and the amount of nicotine transferred into the smoke were studied under different smoking conditions. The combustion temperatures of domestic commercial blended cigarettes were not affected by smoking procedures or amount of moisture in the cigarette. No significant differences in the amount of nicotine transferred into smoke were observed between the cigarettes with medium (10.9%) and high (15.4%) moisture contents, while the values obtained from the low moisture content (6.6%) cigarettes were always slightly higher than those obtained from medium or high moisture content cigarette.     

Stimulation of rapidly adapting receptors in canine lungs by a single breath of cigarette smoke

Inhalation of smoke generated from high-nicotine cigarettes frequently evoked an immediate augmented inspiration in conscious dogs (J. Appl. Physiol. 54: 562-570, 1983); this reflex response was believed to result from a stimulation of rapidly adapting receptors in the lungs. To test this hypothesis, we recorded the vagal afferent activity arising from the rapidly adapting receptors in the lungs and delivered 120 ml of high- and low-nicotine cigarette smoke separately in a single ventilatory cycle in 20 anesthetized open-chest and artificially ventilated dogs. These receptors were stimulated on the first breath of delivery of smoke generated by high-nicotine cigarettes; activity increased from a base line of 0.9 +/- 0.2 to a peak of 9.9 +/- 1.2 (SE) impulses/breath (n = 58). After three to six breaths when the receptors' discharge returned toward base-line activity, a delayed increase of activity emerged (peak activity = 3.4 +/- 0.6 impulses/breath, n = 58) in 32 of the 58 receptors studied and lasted for three to seven breaths. By contrast, only a mild stimulatory effect of low-nicotine cigarette smoke was found, either immediately or after a delay, in 15 of the 54 receptors studied. We conclude that rapidly adapting receptors are stimulated by a single breath of cigarette smoke and that nicotine is the primary stimulant agent.     

Effects of lung volume, bronchoconstriction, and cigarette smoke on morphometric airway dimensions

To examine the role of airway wall thickening in the bronchial hyperresponsiveness observed after exposure to cigarette smoke, we compared the airway dimensions of guinea pigs exposed to smoke (n = 7) or air (n = 7). After exposure the animals were anesthetized with urethan, pulmonary resistance was measured, and the lungs were removed, distended with Formalin, and fixed near functional residual capacity. The effects of lung inflation and bronchoconstriction on airway dimensions were studied separately by distending and fixing lungs with Formalin at total lung capacity (TLC) (n = 3), 50% TLC (n = 3), and 25% TLC (n = 3) or near residual volume after bronchoconstriction (n = 3). On transverse sections of extraparenchymal and intraparenchymal airways the following dimensions were measured: the internal area (Ai) and internal perimeter (Pi), defined by the epithelium, and the external area (Ae) and external perimeter (Pe), defined by the outer border of smooth muscle. Airway wall area (WA) was then calculated, WA = Ae - Ai. Ai, Pe, and Ae decreased with decreasing lung volume and after bronchoconstriction. However, WA and Pi did not change significantly with lung volume or after bronchoconstriction. After cigarette smoke exposure airway resistance was increased (P less than 0.05); however, there was no difference in WA between the smoke- and air-exposed groups when the airways were matched by Pi. We conclude that Pi and WA are constant despite changes in lung volume and smooth muscle tone and that airway hyperresponsiveness induced by cigarette smoke is not mediated by increased airway wall thickness.     

The Pro-Proliferative Effects of Nicotine and Its Underlying Mechanism on Rat Airway Smooth Muscle Cells

Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway.     

Effect of bronchomotor tone on work rate of breathing

We studied the optimal airway caliber for minimizing the work rate of breathing in the lung (W) with different bronchomotor tones in six normal subjects. The inhalation of methacholine contracted airway smooth muscle, and the inhalation of salbutamol relaxed it. To calculate W at a given alveolar ventilation (VA), anatomical dead space (VDanat), pulmonary resistance (RL), and dynamic compliance were measured simultaneously, breath by breath, during various breathing maneuvers. VDanat increased and RL decreased with both increased breathing frequency and tidal volume, even at a given airway tone. This suggests that the airway caliber varied even at a given bronchomotor tone. The minimum W at a given VA increased in constricted airways, but there was no significant difference between control airways after saline inhalation and relaxed airways. It has been suggested that airway smooth muscle tones at both control and relaxed conditions bring W to a minimum and that the airway smooth muscle tone existing in the control state acts to keep the airway caliber optimal in order to minimize the W and stabilize the airway mechanics.     

Dose-response curves of central and peripheral airways to nicotine injections in dogs

The dose-response curves of the central and peripheral airways to intravenously injected nicotine were studied in 55 anesthetized dogs. With intact vagi, nicotine caused a dose-dependent increase in central airway resistance (Rc) similar to the increase in peripheral airway resistance (Rp) at concentrations ranging from 4 to 64 micrograms/kg. However, the responses of both Rc and Rp fell progressively when sequential doses of nicotine greater than 256 micrograms/kg were administered. With intact vagi and the administration of propranolol, there was a greater increase in Rp than in Rc at a nicotine dose of 64 micrograms/kg (P less than 0.05). With vagotomy, the responsiveness of both central and peripheral airways to nicotine decreased with doses of nicotine less than 64 micrograms/kg, but with doses of nicotine greater than 256 micrograms/kg the suppressive effect of nicotine on both Rc and Rp was less than that seen with intact vagi. Under conditions in which the vagi were cut and atropine administered, the responsiveness of nicotine was even further depressed. Combinations either of atropine and chlorpheniramine or atropine and phenoxybenzamine also completely blocked reactions to nicotine. Additionally reactions to nicotine were completely blocked by hexamethonium. These results suggest that nicotine increases both Rc and Rp mainly through a vagal reflex and stimulation of the parasympathetic ganglia.     

Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with transient receptor potential melastatin 8 (TRPM8) ion channels in cold-sensitive sensory neurons, and transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by smoke irritants (acrolein, acetic acid, and cyclohexanone). Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did eucalyptol (460 ppm), another TRPM8 agonist. Menthol's effects were reversed by a TRPM8 antagonist, AMTB. Menthol's effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counterirritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote nicotine addiction and smoking-related morbidities.