INTRODUCTION

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AIRWAY HYPERRESPONSIVENESS and variable airflow obstruction are key features of airway-related diseases such as asthma; indeed, one might say that these are the most clinically relevant features of such airway disorders. For this reason, it is essential to have a good understanding of the mechanisms underlying excitation-contraction coupling in airway smooth muscle (ASM).

Excitation-contraction coupling in cardiac and skeletal muscles, as well as certain types of smooth muscle (vascular and gastrointestinal), involves membrane depolarization, resulting in Ca²⁺ entry via voltage-dependent ("L-type") Ca²⁺ channels. Thus L-type Ca²⁺-channel blockers are very effective in controlling vascular and cardiac contractions in hypertension. In ASM, excitation is also accompanied by membrane depolarization via activation of large inward Cl and nonselective cation currents (14, 36). This depolarization activates voltage-dependent Ca²⁺ current (8, 18), which is sufficient for contraction, as attested to by the robust responses evoked by high millimolar concentrations of KCl (11, 16). Unfortunately, however, Ca²⁺-channel blockers are ineffective in treating bronchoconstriction. Ca²⁺ entry in ASM may also involve nonselective cation channels (7, 36) and/or "receptor-operated" Ca²⁺ channels (22).

The next advance in our understanding of ASM excitation-contraction coupling came with a series of studies showing that spasmogens act by activation of phospholipase C and generation of inositol 1,4,5-trisphosphate (IP₃), which, in turn, triggers release of Ca²⁺ stored within the sarcoplasmic reticulum (31, 38). It is now known that the sarcoplasmic reticulum forms sheets around the periphery of the cytosol, thereby dividing the cytosol into two pools, the peripheral space immediately underneath the plasmalemma where ion channels are found (many of them being regulated by Ca²⁺) and the deep cytosolic space where the contractile apparatus is found, and that intracellular Ca²⁺ concentration ([Ca²⁺]_i) in these two spaces can be regulated very differently (11). There have been numerous studies of the mechanisms underlying Ca²⁺ handling in ASM (2, 11, 15, 16, 29, 38). Surprisingly, substantial ASM contraction can still be evoked under conditions in which internally sequestered Ca²⁺ is not available for contraction. For example, repeated stimulation of Ca²⁺ release in the presence of agents that inhibit the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) leads to complete abrogation of Ca²⁺-dependent Cl currents, suggesting that the store has been functionally depleted, and yet contractions in those same cells are completely unaffected (15)! Others have used contractile responses as an index of [Ca²⁺]_i in ASM (albeit a much less reliable one) and have come to similar conclusions (2). Instead, the voltage-dependent Ca²⁺ channels may play an important role in refilling the sarcoplasmic reticulum (1, 2, 15, 25). Clearly then, other excitation-contraction coupling mechanisms remain to be examined.

Recently, it has come to be appreciated that spasmogens can constrict smooth muscle both by increasing [Ca²⁺]_i and by increasing the sensitivity of the contractile apparatus to Ca²⁺, such that even basal ("resting") levels of [Ca²⁺]_i are sufficient to evoke contraction (or that a given level of excitation leads to a greater level of contraction). Although some suggest that this involves the diacylglycerol-protein kinase C pathway (which is coincident with the IP₃-Ca²⁺-release pathway), direct evidence for this, at least in ASM, is lacking. Instead, many studies point to the role of Rho and Rho kinase, the latter of which phosphorylates myosin light chain phosphatase, leading to accumulation of phosphorylated myosin light chain and thus contraction (3, 4, 26, 32, 39).

A growing body of evidence attests to differences in structure and function as one progresses down the airways, including differential expression of ion conductances, receptors, and functional responses, to name only a few (10, 13, 30). This fact may call into question the value of many studies of ASM physiology, because these have used tracheal tissues and not smaller airways.

During the course of our laboratory's earlier studies of Ca²⁺ handling in ASM (13, 16), we found cyclopiazonic acid (CPA; inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase) to have mixed effects on tone in carbachol (CCh)-stimulated ASM, evoking either a contraction or a relaxation, depending on several factors, including the degree of preconstriction and the type of tissue (tracheal vs. bronchial). In this study, we sought to examine the mechanism(s) underlying 1) the CPA-induced enhancement of tone during modest muscarinic stimulation; 2) the CPA-induced relaxation seen during aggressive muscarinic stimulation; and 3) the tone that persists in the presence of CPA, particularly with a view to understanding the differences between tracheal (TSM) and bronchial smooth muscle (BSM) tissues.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of isolated tissues and single cells. Whole lobes of lung and tracheae were obtained from dogs that had been euthanized using pentobarbital sodium (100 mg/kg). TSM was isolated by removing connective tissue, vasculature, and epithelium, and then was cut into strips parallel to the muscle fibers (~1 mm wide). Lobes of lung were pinned out, the overlying parenchyma and pulmonary vasculature were removed, and ring segments (~4-5 mm long) of fifth- to sixth-order bronchi (outer diameter, 2-6 mm) were excised. For single-cell studies, TSM strips (0.5-1.0 g wet weight) were transferred to dissociation buffer (composition given below) containing collagenase (type IV; 2.7 U/ml), elastase (type IV; 12.5 U/ml), and bovine serum albumin (1 mg/ml) and then were either dissociated immediately or stored at 4°C for dissociation at a later time (<48 h later). We have previously found that cells used immediately and those used after 48 h of refrigeration exhibit similar functional responses (i.e., contraction and activation of Ca²⁺-dependent ion conductances). To dissociate into single TSM cells, tissues in enzyme-containing solution were incubated at 37°C for 60-120 min and then gently triturated. Tissues were either used immediately or stored at 4°C for use the next day; we found no functional differences in tissues that were studied immediately compared with those used after 24-h refrigeration.

Muscle bath technique. Ring segments were mounted into 3-ml muscle baths using stainless steel hooks inserted into the lumen. One hook was fastened to a Grass FT-03 force transducer using silk thread (Ethicon 4-0); the other was attached to a Plexiglas rod, which served as an anchor. Tissues were bathed in Krebs-Ringer buffer (see below for composition) containing indomethacin (10 µM), bubbled with 95% O₂-5% CO₂, and maintained at 37°C; tissues were passively stretched to impose a preload tension of ~1 g (determined to allow maximal responses). Isometric changes in tension were amplified and plotted using a chart recorder or were digitized (2 samples/s) and recorded on-line (DigiMed System Integrator, MicroMed, Louisville, KY) for plotting on the computer. Tissues were equilibrated for 2 h before the experiments were commenced, during which time the tissues were challenged with 60 mM KCl at least once to assess the functional state of each tissue.

Fura 2 fluorimetry. Freshly dissociated cells were studied by using a filter-based photometric system (DeltaScan; Photon Technology International, South Brunswick, NJ). After settling onto a glass coverslip mounted onto a Nikon TMD inverted microscope, cells were loaded with the membrane-permeant form of fura 2 (fura 2-acetoxymethyl ester, 2 µM for 30 min at 37°C) and then superfused continuously with Ringer buffer (2-3 ml/min) at 37°C. Cells were illuminated alternately (0.5 Hz) at the excitation wavelengths and the emitted fluorescence (measured at 510 nm) induced by 340-nm excitation (F₃₄₀), and that induced by 380-nm excitation (F₃₈₀) was measured using a photomultiplier tube assembly. Agonists were applied by pressure ejection from a puffer pipette (Picospritzer II, General Valve, Fairfield, NJ).

Patch-clamp electrophysiology. Single TSM cells were allowed to settle and adhere to the bottom of a recording chamber (1-ml bath volume perfused at 2-3 ml/min) and were studied within 6 h after dissociation. Membrane currents were recorded using the nystatin perforated-patch method, which our laboratory has described in detail previously (10, 11, 16). Briefly, cells were held under voltage clamp at 60 mV using an Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA). Electrodes had a tip resistance of 1-3 M and were filled with an electrode solution containing the following (in mM): 140 KCl, 0.4 CaCl₂, 1 MgCl₂, 1 EGTA, 20 HEPES, pH 7.2, and nystatin (final concentration of 200 µg/ml). Access resistance ranged from 10 to 40 M, and 60-80% series resistance compensation was employed. Data were filtered at 1 kHz, sampled at 2 kHz using pCLAMP6 software (Axon Instruments), and stored on the computer hard drive for later analysis using pCLAMP6 and SigmaPlot software. Corrections were not made for liquid junction potentials [previously found to be only ~2 mV (14)]. Agonists were applied by pressure ejection from a puffer pipette (Picospritzer II; General Valve). The apparatus is designed to hold two different puffer pipettes, and we are sometimes able to replace these with a new one during the course of a voltage-clamp experiment (only when the cell can be lifted up off the coverslip while maintaining a gigaohm seal on the electrode); thus at least two different concentrations of ACh ([ACh]) could be applied to a given cell. For data analysis, we included only cells that were able to respond to 10⁴ M ACh.

Solutions and chemicals. Dissociation buffer contained the following (in mM): 125 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 0.25 EDTA, 10 D-glucose, and 10 L-taurine, pH 7.0. Single cells were studied in Ringer buffer containing the following (in mM): 130 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 20 HEPES, and 10 D-glucose, pH 7.4. Intact tissues were studied using Krebs-Ringer buffer containing the following (in mM): 116 NaCl, 4.2 KCl, 2.5 CaCl₂, 1.6 NaH₂PO₄, 1.2 MgSO₄, 22 NaHCO₃, and 11 D-glucose, bubbled to maintain pH at 7.4. Indomethacin (10 µM) was also added to the latter to prevent generation of cyclooxygenase metabolites of arachidonic acid. Nominally Ca²⁺-free medium was prepared by omitting CaCl₂ and adding EGTA (either 0.1 or 1.0 mM, as indicated).

Data analysis. At the end of the experiments, tissues were dried and weighed; dry weight was used to standardize the mechanical responses. When CPA triggered phasic activity, the magnitude of the CPA-evoked response was taken to be the difference between tone existing before addition of CPA and tone existing at the minima of the oscillations. Responses are reported as means ± SE; n refers to the number of animals. Statistical comparisons were made using paired Student's t-test or ANOVA (with Newman-Keuls post hoc test), as appropriate. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mixed effects of CPA on muscarinic responses. CPA (6 × 10⁵ M) had mixed effects on tone evoked by CCh in canine TSM: it evoked substantial contraction in tissues pretreated with relatively low concentrations of CCh [i.e., 10⁸ M, which elicits ~10% of the maximal contractile response (12)] but dramatic relaxations in tissues exposed to half-maximally effective concentrations of CCh or greater (Fig. 1). Approximately 10-20 min after the addition of CPA, many tracheal tissues exhibited phasic activity, "oscillations" in tone, similar in time course to those that our laboratory has described previously (13). Figure 1B shows the mean peak magnitude of mechanical activity after stimulation with each concentration of CCh, as well as tone existing after the addition of CPA. The CPA-induced increase in tone during low levels of muscarinic stimulation and the CPA-induced decrease in tone during more aggressive muscarinic stimulation were both statistically significant. Interestingly, phasic activity was seen in the majority (20 of 33) of TSM tissues preconstricted with 10⁸-10⁶ M CCh but not in any of 32 tissues constricted with 10⁵-10⁴ M CCh.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Mixed effects of cyclopiazonic acid (CPA) on muscarinic tone. A: representative tracings showing the contractile or relaxant responses evoked by CPA (6 × 105 M) in canine tracheal smooth muscle (TSM) tissues preconstricted to varying degrees using carbachol (CCh; 108-104 M). Mean (±SE) magnitudes (g tension/mg tissue) of responses in TSM (B) and bronchial smooth muscle (BSM) tissues (C) studied in this fashion are given. n = 5 for all. Open bars, tone existing before addition of CPA; solid bars, tone in those same tissues after addition of CPA. * Statistically significant change in muscarinic tone on addition of CPA, P < 0.05.

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View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effects of CPA on muscarinically evoked Ca2+ transients. Representative tracings from canine tracheal myocytes loaded with fura 2 and challenged with CPA alone (6 × 105 M; A) or during stimulation with either 108 CCh (B) or 104 M CCh (C). D: mean (±SE) magnitudes of transients evoked by 108 CCh and then CPA (n = 7) or by 104 M CCh and then CPA (n = 6). F340/F380, ratio of fluorescence induced by 340- to 380-nm excitation; [Ca2+]i, change in intracellular Ca2+ concentration.

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View larger version (18K): [in this window] [in a new window]   Fig. 3.   ACh-evoked membrane currents. A: in 1 cell held under voltage clamp at 60 mV, ACh (1010 M) evoked large inward current; a subsequent application of 104 M ACh 3 min later evoked a response of comparable magnitude. Fraction (%) of cells responding (B) and absolute magnitude of the currents evoked (C) in 23 cells (n = 5) studied in this way are given.

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Mechanism underlying salutary effect of CPA on CCh-evoked contraction. Nelson et al. (17, 23) have shown that CPA (and other agents that release internally sequestered Ca²⁺) increases vascular tone by decreasing Ca²⁺ spark frequency, thereby decreasing Ca²⁺-dependent K⁺-channel activity, leading to membrane depolarization and voltage-dependent Ca²⁺ influx. This mechanism may also account in part for the CPA-evoked enhancement of tone in our tissues. On the other hand, by allowing more Ca²⁺ to accumulate in the subplasmalemmal space, CPA may increase tone by increased activation of Ca²⁺-dependent Cl channels and thus voltage-dependent Ca²⁺ influx. We tested these two possibilities using TEA to block Ca²⁺-dependent K⁺ channels and niflumic acid to block Ca²⁺-dependent Cl channels (Fig. 4). Whereas niflumic acid (10⁴ M) suppressed the contraction evoked by 10⁸ M CCh, subsequent addition of CPA evoked a further eightfold increase in tone (to 802 ± 205% of that evoked by CCh). TEA at 5 mM had no effect on baseline tone or muscarinic tone, and subsequent addition of CPA also still evoked a statistically significant increase (to 178 ± 24%) of tone. At 30 mM, however, TEA markedly increased baseline tone, thereby occluding the subsequent responses to 10⁸ M CCh and to CPA (not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   CPA-evoked enhancement of muscarinic tone. Representative tracings show CPA-induced enhancement of tone evoked by 108 M CCh in tracheal tissues pretreated with niflumic acid (104 M; A) or tetraethylammonium (TEA 5 mM; B).

Mechanism underlying CPA-evoked relaxations. CPA may trigger relaxations by allowing more Ca²⁺ to accumulate in the subplasmalemmal space, leading to increased activation of Ca²⁺-dependent K⁺ channels and membrane hyperpolarization (17, 23). To test this hypothesis, we pretreated tissues with TEA (30 mM; n = 6) to block Ca²⁺-dependent K⁺ channels or with KCl (120 mM; n = 6) to prevent membrane hyperpolarization even if the K⁺ channels were to open, before assessing CPA-evoked relaxations. CPA could still reverse CCh tone under these conditions (Fig. 5), and the magnitudes of those relaxations were not statistically significantly different from the control responses (65 ± 6 vs. 59 ± 4%). However, CPA did not evoke a relaxation in tissues precontracted with 120 mM KCl alone (Fig. 5; n = 6) or in tissues permeabilized using -escin and precontracted directly by Ca²⁺ in the bathing medium (Fig. 5; n = 5). In fact, CPA elevated tone under those circumstances by 5.0 ± 2.5 and 202 ± 80%, respectively. The absence of relaxations in the presence of KCl alone or of -escin indicates that CPA relaxation is not due to some nonspecific effect on the contractile apparatus. We, therefore, conclude that the CPA-evoked relaxation reflects some kind of "Ca²⁺-recycling" phenomenon: that is, sustained CCh-induced release of internally sequestered Ca²⁺ requires ongoing refilling of the sarcoplasmic reticulum to maintain a certain level of tone.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   CPA-evoked reversal of muscarinic tone. Representative tracings illustrate ability of CPA (6 × 105 M) to reverse tone evoked by 104 M CCh, even during exposure to TEA (25 mM; D) or high KCl (120 mM; C) but its inability to reverse tone evoked by KCl alone (120 mM; A) or during permeabilization of the membrane by -escin (104 M; B).

Ca²+-influx pathways. The Ca²⁺-recycling mechanism proposed above would require a certain degree of ongoing Ca²⁺ influx across the membrane to maintain the filling state of the sarcoplasmic reticulum (see DISCUSSION). Others have investigated the extent to which this Ca²⁺ enters via voltage-dependent Ca²⁺ channels (2, 25); we used a number of strategies to examine whether voltage-independent pathways are involved.

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View larger version (24K): [in this window] [in a new window]   Fig. 6.   Ca2+ influx and sustained muscarinic contraction. Contractions were evoked by CCh (104 M) in TSM (A) and BSM (B) tissues, and the mean peak tone (open bars) and tone existing 20 min after addition of CCh (solid bars) were quantified. This was done under control conditions, as well as in nominally Ca2+-free media with either 0.1 or 1.0 mM EGTA to chelate trace Ca2+, or in the presence of Ni2+ (0.1 or 1.0 mM) or La3+ (1.0 mM) as indicated. Values are means ± SE; n = 5 for all. * Significant change in tone over the course of the 20-min stimulation, P < 0.05.

"Pharmacomechanical" coupling in TSM and BSM. Several studies have shown that, whereas electromechanical coupling is important during submaximal stimulation (5, 6), pharmacomechanical coupling mechanism(s) predominates at the other extreme of the cholinergic dose-response relationship (31). The nature of the latter mechanism(s) is still unclear; however, tyrosine kinases and Rho kinase are emerging as important candidate effectors (3, 4, 26, 32, 39). Also, phosphatidylinositol 3-kinase is coupled to muscarinic receptors in ASM (35) and increases Ca²⁺ sensitivity in colonic smooth muscle (37). We, therefore, compared the effects of the nonspecific tyrosine kinase inhibitor genistein (10⁴ M), CPA (10⁵ M), the Rho kinase inhibitor Y-27632 (10⁵ M), and the phosphatidylinositol 3-kinase inhibitor LY-294002 (10⁵ M) on the CCh dose-response relationship in TSM and BSM tissues.

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View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effects of various inhibitors on CCh dose-response relationship. Mean CCh dose-response relationships in canine TSM (left) and BSM (right) pretreated for 20 min with either genistein (104 M; A), genistein plus CPA (105 M; A), Y-27632 (105 M; B), or LY-294002 (105 M; C), after which the CCh dose-response relationship was investigated. Values are means ± SE; n = 5-7 for all. * Statistically significant difference compared with corresponding control response (ANOVA), P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Many have investigated electromechanical coupling in ASM and found it to be important to muscarinic excitation-contraction coupling only during submaximal levels of stimulation. That is, the upper one-half of the concentration-response relationship shows no correlation with membrane depolarization (5) and is essentially unaffected when voltage-dependent Ca²⁺ influx is prevented using L-type Ca²⁺-channel blockers (6) or K⁺-channel openers (2, 28) or directly using voltage-clamp techniques (15). The responses to other spasmogenic autacoids, such as histamine or leukotrienes, may exhibit a different dependence on electromechanical coupling.

Generally speaking then, voltage-independent mechanisms seem to be most important for contraction in ASM. The first such mechanism to be described was the phosphoinositide cascade, resulting in IP₃-induced release of internal Ca²⁺ (38). This Ca²⁺ release is barely discernible in cells challenged with 10⁸ M ACh [a concentration that is sufficient to increase tone (19)] using photometric techniques that measure the average change in [Ca²⁺]_i throughout the entire cell (Fig. 2; Refs. 27, 29, 38). However, we could demonstrate substantial responses even down to 10¹⁰ M ACh using patch-clamp techniques to monitor [Ca²⁺]_i in the subplasmalemmal space (Fig. 3). Interestingly, another group could visualize Ca²⁺ transients in response to 10¹⁰ M ACh using whole cell photometry in serum-deprived canine TSM cells but not cells grown in serum-containing media (21). They also showed that the fraction of cells responding to ACh decreased with decreasing [ACh], consistent with our own data (Fig. 3). Ongoing SERCA activity serves to refill this intracellular Ca²⁺ pool and moderates changes in [Ca²⁺]_i resulting from Ca²⁺ influx across the membrane (11) (see below). Previously, our laboratory showed that CPA causes a slow and modest elevation of [Ca²⁺]_i (~100 nM) in ASM cells at rest (16). When added to cells stimulated with "threshold"" concentrations of agonist, however, CPA elevates [Ca²⁺]_i several hundred nanomolar (Fig. 2). Thus the CPA-induced enhancement of tone seen in tissues during mild stimulation is due to disruption of a "superficial buffer barrier," resulting in a net greater elevation of [Ca²⁺] in the vicinity of the contractile apparatus. During more aggressive muscarinic stimulation, however, Ca²⁺ release likely surpasses Ca²⁺ uptake, and a significant amount of Ca²⁺ would be discharged via the plasmalemmal Ca²⁺ pump. This "loss" of Ca²⁺ would need to be compensated for in some way by Ca²⁺ influx to prevent a progressive depletion of the sarcoplasmic reticulum; such Ca²⁺ recycling would account for the collapse of the muscarinic responses in TSM tissues bathed in the absence of external Ca²⁺ (Fig. 6). Thus the CPA-evoked relaxation might reflect a greater uncompensated ejection of Ca²⁺ from the cell by plasmalemmal Ca²⁺-ATPase activity. Presently, there are no selective blockers for plasmalemmal Ca²⁺-ATPase that would allow us to test this hypothesis.

Thus there is somewhat of a paradox: muscarinic contractions are mediated largely by release of internal Ca²⁺ and yet are dependent to some degree on Ca²⁺ influx. Liu and Farley (19) have also shown the same to be true for maintained Cl-current oscillations during sustained cholinergic stimulation in TSM. In fact, there is accumulating evidence that this may represent an important function of the voltage-dependent Ca²⁺ channels in these tissues (1, 2, 15); when the intracellular Ca²⁺ pool is eliminated by agents such as CPA, the relative importance of voltage-dependent Ca²⁺ influx to excitation-contraction coupling is dramatically increased (33). Other studies suggest that Ca²⁺ may also enter via nonselective cation channels (7, 36), which are activated by cholinergic stimulation (14). Ni²⁺ at millimolar concentrations is expected to be sufficient to block both of these pathways and yet does not mimic the effect of removing external Ca²⁺ (Fig. 6); others have demonstrated refilling of the sarcoplasmic reticulum in ASM via a Ni²⁺-insensitive Ca²⁺-influx pathway (20). La³⁺ did inhibit muscarinic responses (Fig. 6) but may have done so by a number of other nonspecific effects.

Interestingly, contractions in BSM are much less dependent on Ca²⁺ influx: removal of external Ca²⁺ markedly attenuated the responses to CCh in TSM but not in BSM (Fig. 6). Whereas it might be argued that this is related to the differing diffusional barriers in TSM strips vs. BSM rings, we find that the responses to CCh or CPA in both tissues develop rapidly and are easily washed out, suggesting that molecules as large as these can diffuse freely into and out of the tissues. Previously, our laboratory (13) showed that these tissues differ markedly in their responses to CPA under basal conditions (i.e., not when Ca²⁺ release is being enhanced by CCh): BSM tissues respond with a large contraction, whereas TSM tissues exhibited little or no response. These data are consistent with a continuous SERCA-mediated uptake of Ca²⁺ into the sarcoplasmic reticulum (inhibition of which leads to elevation of [Ca²⁺]_i and contraction), which is much more efficient in BSM than in TSM; this hypothesis would also account for the differing sensitivity to Ca²⁺-deficient media between these tissues. These functional differences between TSM and BSM are very important, because it is the bronchial airways that are more sensitive to inflammation and that play a greater role in determining resistance to airflow than the trachealis (24). This calls into question the clinical relevance of studies done using TSM tissues.

Finally, we investigated other putative signaling pathways that might be involved in excitation-contraction coupling, because substantial tone could still be seen in the presence of CPA (Fig. 1); previously, our laboratory found that we could completely deplete the sarcoplasmic reticulum of Ca²⁺ (as indicated by complete loss of Cl currents) and prevent voltage-dependent Ca²⁺ influx using voltage-clamp techniques, and yet contractions in those very same cells were unaffected (15)! Diacylglycerol is also generated concurrently with IP₃ and leads to activation of protein kinase C; however, the importance of this latter pathway to contraction in ASM per se is debated. Other more recently described effector pathways include Rho kinase-mediated suppression of myosin light chain phosphatase (3, 4, 26, 32, 39), extracellular regulated kinase-mediated phosphorylation of caldesmon/calponin (37), phosphorylation of cytoskeletal proteins (9), and tyrosine kinase activity (34). The relative contributions of these and other pathways at different extremes of muscarinic stimulation have not yet been investigated. We found that inhibition of Rho kinase produces a much more profound effect (>75% inhibition) on the lower end of the CCh dose-response relationship than that seen during maximal cholinergic stimulation (<25%) (Fig. 7); submaximal cholinergic contractions are more relevant physiologically and clinically than maximal cholinergic excitation. Tyrosine kinase seems to be important in BSM but not TSM (Fig. 7A); others have found tyrosine kinase to be important for serotonin-induced Ca²⁺ release (34) and for refilling of the Ca²⁺ store (20).

In conclusion, there are several diverse mechanisms underlying excitation-contraction coupling in ASM, the relative importance of which seem to vary, depending on the spasmogen used (muscarinic vs. histamine), the degree of stimulation (threshold vs. maximal), the tissue (trachea vs. bronchi), and possibly also the species. It may be that there is a change in the relative contributions of these pathways during pathological conditions such as airway inflammation and asthma; this change might account in part for the increased airway hyperresponsiveness seen in these clinical conditions.