The intracellular mechanisms involved in the alpha-adrenoceptor- or hyperventilation-induced bronchoconstriction remain unknown. Because there is a direct relationship between phosphatidylinositol (PI) metabolism and airway smooth muscle contraction induced by muscarinic agonists, the authors examined the effects of carbachol (CCh), norepinephrine (NE), and hypocapnia on PI turnover in the airway smooth muscle.
Rat tracheal slices were incubated in Krebs-Henseleit solution containing LiCl and [3H]myo-inositol in the presence of NE, CCh, or neither. The PCO2 in the solution was 36 +/- 3 mmHg (normocapnia), 19 +/- 2 mmHg (moderate hypocapnia), or 5 +/- 2 mmHg (severe hypocapnia), respectively. [3H]inositol monophosphate (IP1) formed was counted with a liquid scintillation counter.
Basal IP1 formed was greater at severe hypocapnia than at normocapnia. Norepinephrine- and CCh-induced IP1 formation were also greater at hypocapnia than at normocapnia.
These results indicate that CCh, NE, and hypocapnia stimulate PI turnover in the airway smooth muscle, which would cause bronchoconstriction, and hypocapnia also augments NE- and CCh-induced PI turnover, which could cause worsening of exercise-induced asthma and vagotonic asthma, respectively.
Key words: Lungs, hyperventilation: hypocapnia. Phosphatidylinositol turnover: inositol monophosphate. Sympathetic nervous system, catecholamines: norepinephrine.
BOTH muscarinic receptors and alpha-adrenoceptors have been shown to exist in airway smooth muscle.  Baron et al.  reported that phosphatidylinositol (PI) metabolism plays a role in the pharmacomechanical coupling of muscarinic receptor-mediated airway smooth muscle contraction. Hashimoto et al. demonstrated that inositol 1,4,5-triphosphate (IP3) may initiate smooth muscle contraction in dogs.  Meurs et al.  demonstrated evidence for a direct relationship between PI metabolism and airway smooth muscle contraction induced by muscarinic agonists. On the other hand, some studies have reported that alpha-adrenoceptor agonists stimulate human airway smooth muscle contraction, [5–7] that alpha-adrenoceptors play a role in exercise-induced bronchoconstriction,  and that plasma norepinephrine (NE) increases in normal and asthmatic subjects during exercise.  However, the intracellular mechanisms involved in the alpha-adrenoceptor-induced bronchoconstriction remain unknown.
It is known that hyperventilation [10–13] provokes bronchoconstriction and worsens exercise-induced asthma. Several investigators reported that bronchoconstriction occurs in asthmatic patients during exercise more readily when they breathe cold dry air than when they breathe warm moist air, and suggested that either heat loss or water loss worsened exercise-induced asthma. [14–18] Thus, Freed et al.  speculated that drying of the bronchial mucosa may inactivate an epithelial-dependent relaxant process and simultaneously stimulate release of bronchoactive mediators from osmo-sensitive cells, and that cooling per se would tend to offset the effect of hyperventilation to provoke bronchoconstriction. On the other hand, hyperventilation could not induce airway obstruction when end-tidal CO sub 2 was maintained at a normal resting level.  Thus, it seems probable that hypocapnia plays an essential role in the genesis of hyperventilation-induced bronchoconstriction. [10–13].
Although both NE and hypocapnia seem to play essential roles in exercise-induced asthma, the mechanisms remain unknown. The current study was designed using rat tracheal slices to clarify whether NE or hypocapnia could stimulate PI turnover, which is an important physiologic step in the bronchoconstriction process.
Materials and Methods
The technique of Brown et al.  was used. Inositol 1,4,5-triphosphate is rapidly degraded into inositol monophosphate (IP sub 1), which is recycled back to phosphatidylinositol (PI) via free inositol. Lithium sup + inhibits the conversion of IP1into inositol. Thus, in the presence of Lithium sup +, the accumulation rate of IP1reflects the extent of PI turnover.  We measured3[H]IP1in tracheal slices incubated with [sup 3 Hydrogen]myo-inositol (Amersham, Tokyo, Japan). The studies were conducted under guidelines approved by the Animal Care Committee of Nagasaki University School of Medicine. Ninety-four male Wistar rats (Charles River, Yokohama, Japan) weighing 250–350 g were used for experiments. The rats were stunned by cervical dislocation and decapitated, and the tracheas were rapidly isolated. For tissue preparation without epithelium, epithelium was removed by rubbing with cotton gauze. Trachea with or without epithelium was cut longitudinally and chopped into 1-mm-wide pieces with a McIlwain tissue chopper (The Mickle Laboratory Engineering, Gomshall, England). Briefly, three pieces of the tracheal slice were placed in small flat-bottomed tubes and preincubated for 15 min in Krebs-Henseleit (K-H) solution (composition in mM: NaCl 118, KCl 4.7, CaCl21.3, KH2PO41.2, MgSO sub 4 1.2, NaHCO325, glucose 10, and Na2-EDTA 0.05) containing 5 mM LiCl. The solution was continuously aerated with 95% O sub 2/5% CO2. An aliquot of 0.5 micro Ci [sup 3 Hydrogen]myo-inositol was then added to each tube (final concentration 0.1 micro Meter in 300 micro liter incubation volume) and the tubes were flushed with 95% O2/5% CO2, capped, set in a shaking bath at 37 degrees Celsius, and incubated for 30 min (time 0).
Effects of Norepinephrine and Carbachol on IP sub 1 Formation
The reaction was started at time 0 when NE, carbachol (CCh), or neither (basal) was added. The tubes were reaerated with 95% O2/5% CO2, recapped, and reincubated for 0, 15, 30, 45, and 60 min. The reaction was stopped with 940 micro liter chloroform:methanol (1:2 v/v). Chloroform and water were then added (310 micro liter each) and the phases were separated by centrifugation with 90 g for 5 min. [sup 3 Hydrogen]IP1was separated from [sup 3 Hydrogen]myo-inositol in the water phase by column chromatography using Dowex AG 1-X8 resin (Bio Rad, Richmond, CA) in the formate form. The “n” refers to the number of experiments and one experiment includes the mean value of duplicate results. The [sup 3 Hydrogen]IP1formed in the tracheal slices was counted with a liquid scintillation counter and presented by disintegration per minute (DPM). The counts in DPM of two samples were averaged and the average DPMs of the blank values (no slices present) were subtracted to obtain the experimental data.
The Effect of Hypocapnia on Monophosphate Formation
The tracheal slices were taken out at time 0, washed, wiped, and put into new K-H solution, containing 0.5 micro Ci [sup 3 Hydrogen]myo-inositol. The conditions of aeration and pH of solution were fourfold, i.e., 95% O2/5% CO2(pH 7.48), 97.5% O2/2.5% CO2(pH 7.84), 100% O2(pH 8.37), or 95% O2/5% CO sub 2 (pH 8.37 titrated with NaOH)(Table 1). The pH and partial pressure of CO2and O2were assayed with an ABL Acid Base Analyzer (Radiometer, Copenhagen, Denmark). The reaction was started by adding NE, CCh, or neither 15 min after putting into the new K-H solution. The tubes (300 micro liter incubation volume) were reaerated, recapped, and reincubated for an additional 45 min. The reaction was stopped with 940 micro liter chloroform:methanol (1:2 v/v), followed by the same procedure described above.
Data were expressed as mean plus/minus SE. The results of repeated measures and multiple groups were analyzed by one-way ANOVA. Multiple pairwise comparisons between groups were assessed by Scheffe's test. A comparison between two groups was assessed by Student's t test. A P value < 0.05 was considered significant.
Time course of IP1formation after adding NE (2.5 micro Meter), CCh (5.5 micro Meter), or neither (basal) are shown in Figure 1. Basal IP1formation reached a level of 168 plus/minus 12 DPM after 60 min and, in the presence of NE or CCh, IP1formed was 252 plus/minus 23 DPM and 615 plus/minus 39 DPM, respectively. The effects of hypocapnia on IP1formation were shown in Figure 2and Figure 3. Basal IP1formation was 150 plus/minus 8 DPM under normocapnia and 245 plus/minus 18 DPM under severe hypocapnia, respectively, and there was a significant difference between normocapnia and severe hypocapnia. Monophosphate formed in the presence of 2.5 micro Meter NE was 272 plus/minus 21 DPM under normocapnia and 356 plus/minus 23 DPM under severe hypocapnia, respectively, and there was a significant difference between normocapnia and severe hypocapnia. Monophosphate formed in the presence of 0.55 micro Meter CCh was 300 plus/minus 10 DPM under normocapnia and 412 plus/minus 25 DPM under severe hypocapnia, respectively, and there was a significant difference between them. Monophosphate formed in the presence of 5.5 micro Meter CCh was not significantly different between normocapnia and hypocapnia. As shown in Figure 4, removal of the epithelium did not influence basal IP1formation under either normocapnia or hypocapnia. Figure 5shows roles of the epithelium in the IP1formation stimulated by NE or CCh. Monophosphate formation stimulated by NE was 315 plus/minus 7 DPM in the presence of epithelium and 535 plus/minus 48 DPM in the absence of epithelium. Thus, removal of the epithelium significantly enhanced NE-stimulated IP1formation. In contrast, IP1formation stimulated by CCh was not influenced by removal of the epithelium. The effects of pH and severe hypocapnia on basal IP1formation were shown in Figure 6. The basal IP1formation was not influenced by an increase in extracellular pH under normocapnia, whereas it was enhanced by severe hypocapnia.
Histochemical analysis of human airways reveals a dense network of parasympathetic fibers.  Acetylcholine released from parasympathetic nerve terminals activates muscarinic receptors in airway smooth muscle cell membrane, and contracts airway smooth muscle. Carbachol was also shown to stimulate IP3formation in animal tracheal smooth muscle,  and the present results also show that CCh stimulates IP1formation. When muscarinic receptors are stimulated to activate the phospholipase C (PLC), phosphatidylinositol-4,5-bisphosphate (PIP2) is hydrolyzed into IP sub 3 and diacylglycerol. Inositol 1,4,5-triphosphate mobilizes Calcium sup ++ from sarcoplasmic reticulum,  whereas diacylglycerol activates protein kinase C (PKC), which may also be a mechanism of modulating or controlling smooth muscle tension.  Subsequently, the increase in cytoplasmic Calcium sup ++ concentration and activation of PKC may cause smooth muscle contraction.
Park and Rasmussen [25,26] have reported that the contractile response of tracheal smooth muscle strips to CCh stimulation reaches the plateau within 2–3 min and is sustained with no loss of tension after many hours of incubation with the agonist. Giembycz and Rodgers  have provided evidence that a rapid, short-lived increase in IP3induced by CCh stimulation precedes the development of tension. Phosphatidylinositol-4,5-bisphosphate, precursor of IP3, formation decreases rapidly and remained at this new steady state level in the continued presence of CCh,  indicating that IP3production is sustained even after a rapid, short-lived increase. Thus, IP3would have an important role for initiating and maintaining contraction of airway smooth muscle. In the current study, we measured the tissue content of IP1as an index of IP3generation, because IP3is rapidly degraded into IP1and the tissue content of IP1increases in a linear manner over 60 min in the presence of CCh.  Wills-Karp  observed both the contraction and the PI response in tracheal tissues of guinea pigs and found that IP1accumulation incubated for 30 min with CCh between 1 micro Meter and 1 mM is between 150 and 250% of basal. Our results show that IP1accumulation for 60 min with 5.5 micro Meter CCh is 370% of basal. The magnitude of IP1accumulation in our study is consistent with their values. Thus, this magnitude of the PI response would be enough to cause the physiologic effect.
Alpha-Adrenoceptors also have been shown to exist in rat airways by autoradiographic analysis.  Catecholamine administration after beta-receptor blockade induces asthma in normal subjects, as well as in patients with asthma.  Although inhalation of prazosin, a specific alpha1-adrenergic antagonist, had little effect on resting airway tone in asthmatics, it partially inhibited exercise-induced asthma in asthmatic subjects. [32,33] Barnes et al. demonstrated that the plasma concentration of NE increases in normal and asthmatic subjects during exercise  and it is considered probable that NE released during exercise would play a significant role in causing exercise-induced asthma. The current results indicate that the stimulation of PI turnover through alpha1-adrenoceptor activation would be the mechanism involved in the NE-induced bronchoconstriction during exercise. We have also examined the roles of epithelium in the NE- or CCh-induced PI turnover. The results show that NE-induced PI turnover is enhanced in the absence of epithelium, whereas CCh-induced PI turnover is not influenced. Farmer et al.  reported that epithelium removal enhances the sensitivity of guinea-pig isolated trachea to the bronchodilator, isoproterenol, and they have indicated that airway epithelium would play a significant role in the uptake and metabolism of catecholamines. Our results would also support this mechanism, and indicate that exercise-induced asthma may occur easily in patients who have the airway epithelium damaged by inflammation.
Airway smooth muscle contraction cannot be induced by hyperventilation if end-tidal CO2is maintained at a normal resting level.  It has been considered possible that hypocapnia plays an essential role in the genesis of hyperventilation-induced bronchoconstriction. [10–13] The current results indicate that stimulation of PI turnover in the airway smooth muscle may be the mechanism involved in the hypocapnia-induced bronchoconstriction. The results also show that hypocapnia has the effects to enhance CCh- and NE-induced IP1formation, indicating that hypocapnia could potentiate both vagotonic asthma and exercise-induced asthma through stimulation of PI turnover.
The mechanism involved in the stimulation by hypocapnia of PI turnover is not clear. Sterling  reported that hypocapnic bronchoconstriction is mediated mainly by cholinergic nerves, because the effect is significantly lessened by atropine. However, hypocapnia (P sub CO2less than 14 mmHg) causes bronchoconstriction that cannot be prevented by atropine. .
The pH of K-H solution is dependent on PCO2, and severe hypocapnia (PCO25 mmHg) makes pH 8.37. To examine whether hypocapnia itself or elevated pH enhances the PI turnover, normocapnic solution was adjusted at pH 8.37 using NaOH. The results show that severe hypocapnia at pH 8.37 enhances PI turnover, whereas normocapnia at pH 8.37 does not affect PI turnover, indicating that hypocapnia itself has the enhancing effect. In contrast to Hydrogen sup +, which is transported by ion exchange systems, CO2diffuses easily through cell membranes. Thus, hypocapnia rapidly produces the elevation of intracellular pH. Schwertz et al.  reported that optimal pH of PLC was between 5.5 and 6.8, whereas Irvine et al.  observed that optimal pH was 5.5–6.8 and 7.5–8.0. Therefore, hypocapnia may activate PLC, and may stimulate PI turnover by increasing intracellular pH to 7.5–8.0.
In conclusion, CCh, NE, and hypocapnia stimulate PI turnover in the airway smooth muscle, which causes bronchoconstriction. Hypocapnia also augments NE- and CCh-induced PI turnover, which may cause worsening of exercise-induced asthma and vagotonic asthma, respectively.