Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

Abstract

1. In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca²⁺-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction.

2. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca²⁺-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca²⁺-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632.

3. Whereas treatment with Y-27632 (1 μM) significantly decreased potency (pEC₅₀) for all agonists, maximal contraction (E_max) was reduced by 23.4±2.8 and 50.4±7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, E_max of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard.

4. Pilocarpine and McN-A-343 induced a very small Ca²⁺-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca²⁺-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca²⁺-sensitization.

5. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca²⁺-mobilization and Ca²⁺-influx for these agonists.

Introduction

Muscarinic receptor stimulation in airway smooth muscle (ASM) results in the activation of phospholipase C, subsequently followed by the production of sn-1,2-diacylglycerol and inositol1,4,5-trisphosphate (IP₃) (Takuwa et al., 1986; Meurs et al., 1989; Yang et al., 1991). In response to IP₃, Ca²⁺ is being mobilized from intracellular stores (Hashimoto et al., 1985), causing a rapid, transient rise in intracellular Ca²⁺-concentration [Ca²⁺]_i (Yang et al., 1993), which is followed by a sustained influx of extracellular Ca²⁺. Smooth muscle contraction is then initiated through the formation of Ca²⁺-calmodulin and subsequent activation of myosin light chain kinase (MLCK), resulting in the phosphorylation of the 20 kDa regulatory myosin light chain (MLC₂₀) (de Lanerolle & Paul, 1991; Pfitzer, 2001). Recently, it has been established that contractile stimuli do not exert their effects only by increasing [Ca²⁺]_i, but also by increasing Ca²⁺-sensitivity of the smooth muscle. One of the main regulators involved in this so-called Ca²⁺-sensitization is Rho-kinase, which acts through inhibition of myosin light chain phosphatase, resulting in an enhanced MLC₂₀ phosphorylation and thus an increased level of contraction at a certain [Ca²⁺]_i (Fukata et al., 2001; Somlyo & Somlyo, 2003).

Contraction of ASM preparations by muscarinic receptor agonists is mediated primarily through M₃-receptor stimulation. Several studies have demonstrated that the involvement of the M₂-receptor in ASM contraction is minor or negligible (Roffel et al., 1988; 1990; Watson et al., 1995). Meurs et al. (1988) compared concentration–response curves (CRCs) for contraction and inositol phosphate accumulation in response to partial and full muscarinic receptor agonists, and demonstrated that a strong linear correlation between these two parameters exists. In addition, they showed a considerable reserve of inositol phosphate production for the full agonists methacholine and oxotremorine, but not for the partial agonist McN-A-343 (Meurs et al., 1988). This transduction reserve is in accordance with studies showing that acetylcholine induces maximal force development by only occupying 4% of the available muscarinic receptors (large receptor reserve), whereas McN-A-343 has to occupy 80% of the receptors (low receptor reserve) to achieve the same degree of force (Gunst et al., 1987; 1989). Furthermore, it has been demonstrated that canine tracheal smooth muscle shortens at a significantly faster rate when contracted with acetylcholine than with McN-A-343 (Gunst et al., 1992). Large differences between full and partial M₃-agonists regarding Ca²⁺-mobilizing capacity have been described, whereas no differences in dependency on Ca²⁺-influx through voltage-dependent channels appear to be present (al-Hassani et al., 1993). To further elucidate mechanisms underlying the differences between full and partial M₃-receptor agonists, we investigated the contribution of Rho-kinase to ASM-contraction, Ca²⁺-mobilization and Ca²⁺-influx in response to the full muscarinic agonist methacholine and the partial muscarinic agonists pilocarpine and McN-A-343 (Meurs et al., 1988; Offer et al., 1991). We demonstrate that these agonists are differentially dependent on Ca²⁺-mobilization/-influx and Rho-kinase for their contractile effects, and that the functional dependency on Ca²⁺-mobilization and -influx is inversely correlated with Rho-kinase dependency.

Methods

Tissue preparation and organ-culture procedure

Bovine tracheae were obtained from local slaughterhouses and rapidly transported to the laboratory in Krebs–Henseleit (KH) buffer of the following composition (mM): NaCl 117.5, KCl 5.60, MgSO₄ 1.18, CaCl₂ 2.50, NaH₂PO₄ 1.28, NaHCO₃ 25.00 and glucose 5.50, pregassed with 5% CO₂ and 95% O₂, pH 7.4. After dissection of the smooth muscle layer and careful removal of mucosa and connective tissue, tracheal smooth muscle strips were prepared while incubated in gassed KH-buffer at room temperature. Care was taken to cut tissue strips with macroscopically identical length (1 cm) and width (2 mm). Tissue strips were washed once in sterile Dulbecco's modification of Eagle's medium (DMEM), supplemented with NaHCO₃ (10 mM), HEPES (20 mM), sodium pyruvate (1 mM), nonessential amino acid mixture (1:100), gentamicin (45 μg ml⁻¹), penicillin (100 U ml⁻¹), streptomycin (100 μg ml⁻¹) and amphotericin B (1.5 μg ml⁻¹). Next, tissue strips were transferred into suspension culture flasks (containing 2.5 ml medium per tissue strip) and maintained overnight.

Isometric tension measurements

Tissue strips, collected from suspension culture flasks, were washed with several volumes of KH-buffer pregassed with 5% CO₂ and 95% O₂, pH 7.4, at 37°C. Subsequently, strips were mounted for isometric recording (Grass force–displacement transducer FT03) in 20 ml water-jacked organ baths, containing KH-buffer at 37°C, continuously gassed with 5% CO₂ and 95% O₂, pH 7.4. During a 90 min equilibration period, with washouts every 30 min, resting tension was gradually adjusted to 3 g. Subsequently, muscle strips were precontracted with 20 and 40 mM isotonic KCl solutions. Following two wash-outs, maximal relaxation was established by the addition of 0.1 μM (−)-isoprenaline. In over 95% of the experiments, no basal myogenic tone was detected. Tension was now re-adjusted to 3 g, immediately followed by two changes of fresh KH-buffer. After another equilibration period of 30 min, cumulative CRCs were constructed to stepwise increasing concentrations of methacholine, pilocarpine or McN-A-343 (1 nM–100 μM). When maximal agonist-induced tension was obtained, the strips were washed several times and maximal relaxation was established using (−)-isoprenaline (10 μM). When used, the Rho-kinase inhibitor Y-27632 (1 μM) was applied to the organ bath 30 min before agonist addition. This concentration has been shown to be effective and selective in smooth muscle (Uehata et al., 1997; Gosens et al., 2004b; Schaafsma et al., 2004). In a separate set of experiments, 100 μM of the alkylating muscarinic receptor antagonist propylbenzilylcholine mustard (PrBCm) was preincubated for 15 min, followed by several washouts, prior to the construction of a cumulative CRC of methacholine.

Isolation of bovine tracheal smooth muscle (BTSM) cells

After the removal of mucosa and connective tissue, tracheal smooth muscle was chopped using a McIlwain tissue chopper, two times at the size of 300 μm and three times at 100 μm. Tissue fragments were washed under sterile conditions (three times) and maintained overnight in DMEM supplemented with NaHCO₃ (10 mM), HEPES (20 mM), penicillin (100 U ml⁻¹), streptomycin (100 μg ml⁻¹) and 10% fetal bovine serum (FBS) in an incubator shaker (Innova 4000) at 37°C, 55 r.p.m.

Tissue fragments were washed three times in Krebs–Ringer–Henseleit (KRH) buffer containing (mM): NaCl 125.0, KCl 6.0, MgCl₂ 2.5, CaCl₂ 1.2, NaH₂PO₄ 1.2, NaHCO₃ 25.0, glucose 11.0, HEPES 25.0, pH 7.4, supplemented with 2.0 mM (±)-dithiothreitol (DTT) and resuspended in a digestion mixture of collagenase P (0.75 mg ml⁻¹), papain (1 mg ml⁻¹) and trypsin inhibitor (1 mg ml⁻¹) in KHR buffer. The suspension was incubated for 20 min at 37°C (55 r.p.m.) and then gently dispersed with a wide-bored pipette. After another 10 min of incubation at 37°C (70 r.p.m.), the suspension was gently dispersed again and filtered over a 50 μm gauze. The cells were collected by centrifugation (1000  × g, 10 min), washed three times with KHR, pH 7.4, supplemented with 2 mg ml⁻¹ FBS (KRH/FBS), and were allowed to regenerate for 1 h at 37°C (55 r.p.m.). Subsequently, the cells were incubated with the fluorescent dye Fura-2/AM (3 μM) for 30 min at 37°C (55 r.p.m.). The loaded cells were washed three times in KRH/FBS and resuspended to a density of 1 × 10⁶ cells ml⁻¹. The cells were kept at room temperature on a Rock-N-Roller (Breda Scientific, The Netherlands) and used within 2–4 h, during which they remained viable and responsive.

Intracellular Ca²⁺-measurements

Fura-2 fluorescence of the cells (excitation wavelengths: 340 and 380 nm; emission wavelength: 510 nm) was measured at 37°C with a Perkin-Elmer spectometer (LS-50B). Each cuvet contained 2 ml of magnetically stirred cell suspension. Ca²⁺-mobilization and -influx induced by methacholine (30 nM–300 μM), pilocarpine (100 nM–300 μM) or McN-A-343 (100 nM–300 μM), added at t=60 s, were measured. When used, Y-27632 (1 μM) was added 30 min prior to agonist addition. The intracellular Ca²⁺-concentration ([Ca²⁺]_i) was calculated every 0.2 s according to Grynkiewicz et al. (1985). At the end of the experiment, the maximal fluorescence ratio (R_max) was determined after adding 0.01% of Triton-X-100 as a permeabilizing agent. The minimal fluorescence ratio (R_min) was determined by addition of 5 mM EGTA to the permeabilized cells.

Data analysis

All data represent means±s.e.m. from n separate experiments. CRCs of contractile responses were analyzed by measuring myogenic tension. No corrections were made for basal tone. Maximal tension (E_max) and pEC₅₀ were calculated from the CRCs. Curves were fitted using the logistic four-parameter model (Sigmaplot 9.0, SPSS Inc.).

Agonist-induced transient rise of [Ca²⁺]_i, representing Ca²⁺-mobilization from internal stores, was expressed as the maximal increase above basal after addition of the agonist. The plateau level of [Ca²⁺]_i, representing Ca²⁺-influx, was measured 2 min after agonist addition and expressed as [Ca²⁺]_i above basal. CRCs for Ca²⁺-mobilization and -influx were expressed as a percentage of the maximal methacholine-induced Ca²⁺-mobilization.

Statistical significance of differences between data was determined using the two-tailed Student's t-test for paired observations or one-way analysis of variance, where appropriate. Differences were considered to be statistically significant when P<0.05.

Materials

Dulbecco's modification of Eagle's Medium (DMEM) and methacholine hydrochloride were obtained from ICN Biomedicals (Costa Mesa, CA, U.S.A.). Foetal bovine serum, NaHCO₃ solution (7.5%), HEPES solution (1 M), sodium pyruvate solution (100 mM), nonessential amino acid mixture, gentamycin solution (10 mg ml⁻¹), penicillin/streptomycin solution (5000 U ml⁻¹; 5000 μg ml⁻¹) and amphotericin B solution (250 μg ml⁻¹) (Fungizone) were obtained from Gibco BRL Life Technologies (Paisley, U.K.). Dithiothreitol (DTT), soybean trypsin inhibitor, Fura-2/AM, pilocarpine hydrochloride and (−)-isoprenaline hydrochloride were from Sigma Chemical Co. (St Louis, MO, U.S.A.). McN-A-343 was purchased from RBI, Natick, MA, U.S.A.

(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632) was obtained from Tocris Cookson Ltd (Bristol, U.K.). L(+)ascorbic acid was from Merck (Darmstadt, Germany). Propylbenzilylcholine mustard was from NEN products (Boston, MA, U.S.A.). Papain and Collagenase P were from Boehringer (Mannheim, Germany). All other chemicals were of analytical grade.

Results

Effects of Rho-kinase inhibition on methacholine-, pilocarpine- and McN-A-343-induced ASM contraction

In BTSM strip preparations, pilocarpine and McN-A-343 reached up to 87 and 84%, respectively, of the maximal contraction (E_max) induced by methacholine, indicating that both pilocarpine and McN-A-343 are partial agonists (Figure 1, Table 1).

Figure 1.

Effects of Rho-kinase inhibition on contraction induced by full and partial M₃-receptor agonists. Methacholine (MCh, a)-, pilocarpine (b)- and McN-A-343 (c)-induced contraction in the absence (open circles) and presence (closed circles) of 1 μM Y-27632 of BTSM strips. Data represent means±s.e.m. of 3–7 experiments, each performed in duplicate.

Table 1.

Effects of Rho-kinase inhibition on contractile properties of bovine tracheal smooth muscle preparations following administration of muscarinic receptor agonists

	Vehicle		Y-27632
	pEC50 (−log M)	Emax (g)	pEC50 (−log M)	Emax (g)
Methacholine	6.73±0.15	25.4±2.5	6.19±0.15**	24.2±2.4
after PrBCm	3.59±0.14	21.8±1.2	2.91±0.17*	16.3±1.6*
Pilocarpine	6.52±0.08	22.2±3.6	6.09±0.02*	17.1±3.1*
McN-A-343	5.26±0.09	21.3±0.2	4.93±0.04*	10.5±1.7*

Data represent means±s.e.m. of 3–7 experiments, each performed in duplicate.

^*P<0.05

^**P<0.01 vs vehicle-treated.

To determine the contribution of Rho-kinase, we used the selective Rho-kinase inhibitor Y-27632 (1 μM). With methacholine, no effect of Rho-kinase inhibition was observed on E_max, but a significant decrease in potency (pEC₅₀) was observed. Interestingly, treatment with Y-27632 significantly decreased E_max of pilocarpine and McN-A-343 by 23.4±2.8 and 50.4±7.9%, respectively, and also significantly decreased potency (Figure 1, Table 1). Remarkably, the effect of Y-27632 in decreasing pEC₅₀ values diminished in the order methacholine>pilocarpine>McN-A-343.

Effects of Rho-kinase inhibition on intracellular Ca²⁺ homeostasis in response to methacholine, pilocarpine and McN-A-343

Ca²⁺-mobilization and -influx were determined in response to several concentrations of methacholine, pilocarpine and McN-A-343 (Figure 2). Both mobilization and influx were calculated as a percentage of the maximal methacholine-induced Ca²⁺-mobilization, which amounted to 349.7±53.9 nM Ca²⁺. Mobilization induced by pilocarpine and McN-A-343 (Figure 2a) was significantly lower as compared to that induced by methacholine, and reached up to only 10.0±0.8 and 5.8±1.6%, respectively (P<0.001, both). Ca²⁺-influx in response to these agonists was also significantly lower (8.9±1.1 and 7.1±1.7%, respectively; Figure 2b) as compared to methacholine-induced influx (13.9±0.5%; P<0.01, both).

Figure 2.

Ca²⁺-mobilization and -influx in response to full and partial M₃-receptor agonists. Methacholine (circles)-, pilocarpine (squares)- and McN-A-343 (triangles)-induced Ca²⁺-mobilization (a) and -influx (b) in BTSM cells. Data are expressed as percentage of maximal Ca²⁺-mobilization induced by methacholine and represent means±s.e.m. of three experiments, each performed in duplicate.

Next, to determine whether the effects of Rho-kinase inhibition on contraction (Figure 1) could be linked to the effects on Ca²⁺-mobilization or -influx, we measured Ca²⁺-responses induced by 100 μM methacholine, pilocarpine and McN-A-343 in the presence and absence of 1 μM Y-27632. As shown in Figure 3, Y-27632 had no inhibitory effect on Ca²⁺-mobilization or -influx induced by either agonist, indicating that the inhibitory effects of Y-27632 on maximal pilocarpine- and McN-A-343-induced contractions are not caused by changes in Ca²⁺-mobilization or -influx.

Figure 3.

Effects of Rho-kinase inhibition on Ca²⁺-responses induced by full and partial M₃-receptor agonists. Ca²⁺-mobilization (a) and -influx (b) in response to methacholine (MCh), pilocarpine and McN-A-343 in the absence (white bars) and presence (gray bars) of 1 μM Y-27632 in BTSM cells. Data are expressed as percentage of maximal Ca²⁺-mobilization induced by methacholine and represent means±s.e.m. of three experiments, each performed in duplicate.

Effects of muscarinic receptor alkylation on the Rho-kinase dependency of the methacholine-induced contraction

To determine whether differences in receptor reserve could be responsible for the difference in Rho-kinase dependency of the contraction induced by full and partial agonists, we removed receptor reserve of the full agonist methacholine by using the alkylating agent propylbenzilylcholine mustard (PrBCm). In contrast to methacholine-induced contraction of untreated strips (Figure 1a), maximal methacholine-induced contraction was Rho-kinase dependent after treatment with PrBCm (24.8±9.2% inhibition of E_max in the presence of Y-27632; P<0.05; Figure 4). These results suggest that the contribution of Rho-kinase to muscarinic agonist-induced contraction is dependent on receptor/transduction reserve. Interestingly, after PrBCm pretreatment, the reduction of the pEC₅₀ value of methacholine by Y-27632 was of similar magnitude as in nonalkylated preparations (Table 1).

Figure 4.

Effects of Rho-kinase inhibition on methacholine-induced contraction after pretreatment with propylbenzilylcholine mustard (PrBCm). Methacholine (MCh)-induced contraction in the absence (closed circles) and presence (triangles) of 1 μM Y-27632 of PrBCm-treated BTSM strips. Data represent means±s.e.m. of 3–4 experiments, each performed in duplicate.

Relationship between Rho-kinase dependency and Ca²⁺-mobilization and -influx

For several agonist concentrations, we have plotted the effectiveness of Y-27632 in decreasing contraction (% inhibition) against the induced Ca²⁺-mobilization and the Ca²⁺-influx (Figure 5). An inverse relationship between Rho-kinase dependency and Ca²⁺-mobilization (Figure 5a) as well as Ca²⁺-influx (Figure 5b) was found.

Figure 5.

Relationship between methacholine (circles)-, pilocarpine (squares)- and McN-A-343 (triangles)-induced Ca²⁺-mobilization (a) and -influx (b) in BTSM cells and Rho-kinase dependency of methacholine-, pilocarpine- and McN-A-343-induced contraction of BTSM strips. Data represent means±s.e.m. of 3–7 experiments, each performed in duplicate.

Discussion

In the present study, we demonstrate that full and partial muscarinic agonists are differentially dependent on Rho-kinase and Ca²⁺-mobilization for their contractile effects. Moreover, we show that there is an inverse (exponential) relationship between the Rho-kinase dependency of the contraction and the Ca²⁺-mobilization as well as the Ca²⁺-influx induced by different concentrations of the agonists. These findings correspond to observations in canine tracheal smooth muscle using acetylcholine and McN-A-343, indicating that differences in Ca²⁺-kinetics are possibly a consequence of differences in potency of muscarinic agonists to activate the same subcellular pathways, rather than from the activation of distinct subcellular mechanisms (al-Hassani et al., 1993).

A receptor-dependent role of Rho-kinase in agonist-induced ASM contraction appears to exist. Both in guinea-pig (Schaafsma et al., 2005) and human (Gosens et al., 2004a) ASM, it was found that growth factors, which are coupled to receptor tyrosine kinases, induce contractions which are fully Rho-kinase dependent, whereas contractions elicited by histamine, which are mediated through G_q/11-coupled H₁ receptors, are not (Gosens et al., 2004a; Schaafsma et al., 2004). In addition, potency and maximal contraction induced by PGF_2α are governed by Rho-kinase activity in guinea-pig tracheal smooth muscle to an important extent, whereas histamine-induced contractions are not (Schaafsma et al., 2004). The agonists used in the present study all function through activation of the M₃-receptor in order to induce ASM contraction. The observed differential involvement of Rho-kinase by partial and full agonists therefore implies that there is not only a receptor-dependent, but also an agonist-dependent role for the participation of Rho-kinase in ASM contraction.

Rho-kinase inhibition resulted in a decrease in potency of methacholine, pilocarpine and McN-A-343, whereas E_max was affected only for pilocarpine and McN-A-343. Remarkably, the strongest decrease in potency was observed for methacholine and the smallest for McN-A-343; in contrast to the effects on E_max, which were absent for methacholine and most pronounced for McN-A-343. This indicates that methacholine is only dependent on Rho-kinase for its contractile effects in the lower concentration range, without requiring Rho-kinase to achieve its maximal contractile response. In BTSM, there is a considerable reserve of inositol phosphate production (transduction reserve) for methacholine, but not for McN-A-343 (Meurs et al., 1988). If the maximally stimulated M₃-receptor generates more Rho-kinase activity than ‘required' for maximal contractile effect, inhibition of this signal transduction will affect the potency, not the efficacy, of the agonist. In other words, methacholine induces the largest IP₃-dependent Ca²⁺-mobilization and -influx, which is probably sufficient to achieve maximal contraction independent of Rho-kinase-mediated Ca²⁺-sensitization. In contrast, very low levels of Ca²⁺-mobilization are observed in response to pilocarpine and McN-A-343, presumably as a consequence of a small and neglectable transduction reserve, respectively (Meurs et al., 1988), and therefore these agonists do rely on Rho-kinase for their maximal contractile response. Also, fully in line with this interpretation, maximal methacholine-induced contraction becomes Rho-kinase dependent after pretreatment with the irreversible muscarinic receptor antagonist PrBCm, demonstrating that the contribution of Rho-kinase to muscarinic agonist-induced contraction is dependent on transductional reserve. Remarkably, in the presence of Y-27632, a similar decrease of potency is observed in PrBCm-treated strips as in untreated strips. An additional explanation for the differences between full and partial agonists in the modulation of the maximal contraction might therefore be that partial agonists have different relative activation profiles for G_q- and G_i/o-proteins as compared to full agonists, as demonstrated in Chinese hamster ovary cells (Akam et al., 2001).

The inverse relationship between Rho-kinase dependency and both Ca²⁺-mobilization and Ca²⁺-influx, as shown in Figure 5, might suggest that Y-27632 is capable of reducing these Ca²⁺-responses. However, we demonstrate that Y-27632 does not inhibit Ca²⁺-mobilization or Ca²⁺-influx for all three agonists at their maximal concentration. In addition, no effect of Rho-kinase inhibition was observed when a submaximal concentration of methacholine (1 μM) was applied (data not shown), corresponding to findings in guinea-pig trachealis, which showed no effects of Y-27632 (1–10 μM) on methacholine (1 μM)-induced Ca²⁺-mobilization (Ito et al., 2001). This strongly suggests that the effects of Rho-kinase inhibition are on the level of Ca²⁺-sensitization rather than on Ca²⁺-mobilization or -influx.

In conclusion, this study shows that full and partial muscarinic receptor agonists are differentially dependent on Rho-kinase for their contractile effects, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Furthermore, we demonstrate that there is an inverse relationship between both Ca²⁺-mobilization and -influx and the functional Rho-kinase dependency, presumably as a consequence of differences in transduction reserve between full and partial M₃-receptor agonists. Moreover, Ca²⁺-mobilization and -influx in response to M₃-receptor stimulation seem to be independent of Rho-kinase, suggesting that differences in functional Rho-kinase dependency are on the level of Ca²⁺-sensitization rather than on Ca²⁺-mobilization or -influx.

Abbreviations

ASM

airway smooth muscle

BTSM

bovine tracheal smooth muscle

CCRC

cumulative concentration–response curve

KH

Krebs–Henseleit

MLC

myosin light chain

MLCK

myosin light chain kinase

MLCP

myosin light chain phosphatase

pEC₅₀

−log₁₀ of the concentration causing 50% of the effect