Role of the cytoskeleton in the regulation of Cl− channels in human embryonic skeletal muscle cells

The effects of volume change and cytoskeleton manipulation on the Cl^– channels in human embryonic skeletal muscle cells were studied. Trypsination, used for production of myoballs, changes the channel properties only a little. When the external osmolarity was reduced from 300 to 270 mosmol/l, the specific Cl^– conductance,g _Cl, (at &-80 mV) of myoballs increased from 5.1±1.9 to 30.4±12.2 S/cm² (SD;n=6) within 15 min. Concomitantly, the kinetics of Cl^– currents, elicited by clamping the membrane potential from a negative to positive values, changed from activation and subsequent slow inactivation to instantaneous activation with fast inactivation. G protein activation, protein kinase action or [Ca²⁺]_i elevation seemed not to be involved in these effects. Similar changes were produced in the absence of a transmembrane osmotic gradient by 500 nM intracellular cytochalasin D (g _Cl=34.3 ±10.3 S/cm²;n=6) or 12.5 M colchicine (g _Cl= 15.4±1.4 S/cm²;n=5). When the external osmolarity was increased to 418 mosmol/l, 1 M cytochalasin D did not affectg _Cl. In four of six cell-attached patches the open probability of the intermediate Cl^– channel was increased after reduction of the bath osmolarity. In inside-out patches, the drugs increased the open probability of the channels. It is concluded that the Cl^– channels are under control of the cytoskeleton.     

Pharmacological block of Ca2+-activated Cl− current in rat vascular smooth muscle cells in short-term primary culture

Ca²⁺-activated Cl^– currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl^– currents can be activated separately by Ca²⁺ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The effects of several Cl^– channel blockers and of spironolactone (a substance known to reduce internal Ca²⁺ loading) on both Cl^– and Ca²⁺ currents were examined. Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2-dicarboxylic acid (DPC) inhibited the Ca²⁺-activated Cl^– current (IC₅₀ values between 16.5 and 306 M) with no effects on the inward Ca²⁺ current and on internal Ca²⁺ loading (tested by measuring the Ca²⁺-activated K⁺ current). These results indicate that the inhibition of Cl^– current by these compounds is due to a direct interaction with the Cl^– channel. In contrast, spironolactone inhibited both K⁺ and Cl^– currents (IC₅₀=7.6 M) by reducing the amount of Ca²⁺ located in the internal stores, whereas the Cl^– current activated by Ca²⁺ current through T-type Ca²⁺ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl^– channels or intracellular Ca²⁺ stores.     

cAMP-dependent activation of small-conductance Cl− channels in HT29 colon carcinoma cells

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT₂₉ and the activation of Cl^– channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was –56±0.4 mV (n=294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K⁺ conductance and a smaller Cl^– conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K⁺ conductance was reversibly inhibited by 1–5 mmol/l Ba²⁺. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl^– conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n=10) and isoproterenol (1 umol/l, n=12) depolarized the cells dose-dependently and reversibly by 12±2 mV and 13±2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl^– conductance. Simultaneously the conductance of the cellattached membrane increased from 155±31 pS to 253±40 pS (VIP, n=4) and from 170±43 pS to 268±56 pS (isoproterenol, n=11), reflecting the gating of Cl^– channels in the cell-attached membrane. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) was without significant effects in resting and in forskolin-stimulated HT₂₉ cells. The agonist-induced conductance increase of the cell-attached nystatin patches was not paralleled by the appearance of detectable single-channel events in these membranes. These data suggest activation of small, non-resolvable Cl^– channels by cAMP.Supported by DFG Gr 480/10 and BMFT 01 GA 88/6     

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.     

Ca-dependent K channels in smooth muscle cells permeabilized by β-escin recorded using the cell-attached patch-clamp technique

Using the cell-attached patch-clamp technique, the activity of single, Ca-dependent K channels was recorded in single smooth muscle cells permeabilized by -escin. The conductance and the relationship between the open probability of the channels and pCa recorded in permeabilized cells were very similar to those obtained in excised inside-out patches. At pCa 7, application of 30 M acetylcholine (ACh) or 0.1 M substance P (SP) together with 1 mM guanosine 5-trisphosphate to permeabilized cells elicited transient bursts of channel openings similar to those which occur in intact cells. Transient activation was also observed when 2–30 M inositol trisphosphate (IP₃) was applied to permeabilized cells. This single channel activity was inhibited by pretreatment with low-molecular-weight heparin at 50–100 g/ml. Channel activity at pCa 7.0 was greatly enhanced by 200 M cyclic adenosine monophosphate. These results provide direct evidence that single Ca-dependent K channel activity is regulated by the transmitters ACh and SP, as well as a second messenger, IP₃, via the release of intracellular Ca from intracellular sites which are blocked by heparin. This novel approach is valuable in elucidating second messenger mechanisms involved in the regulation of single channel activity by transmitters and autacoids, since permeabilization by -escin preserves the entire system of receptor-operated signal transduction and allows intracellular application of second messengers at fixed concentrations.     

Distribution of cGMP-dependent and cGMP-independent Ca2+-activated Cl− conductances in smooth muscle cells from different vascular beds and colon

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca²⁺-activated Cl^– conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl^– conductance has properties distinct from those of the classical Ca²⁺-activated Cl^– conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca²⁺-activated Cl^– current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The classical Ca²⁺-activated Cl^– conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

The outwardly rectifying Cl− channel is not involved in cAMP-mediated Cl− secretion in HT-29 cells: evidence for a very-low-conductance Cl− channel

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl^– channel (ORDIC) during cAMP-mediated Cl^– secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl^– channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4-dinitro-2,2-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ORDIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl^– secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC₅₀=0.5±0.3 mol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl^– secretion unless high concentrations were used (IC₅₀=240±10 mol/l). In order to exclude effects of NPPB other than on the apical Cl^– channel, trans-epithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl^– gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl^– current showed a very-low-frequency Lorentzian noise component (f _c=1.4±0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC. As revealed from fura-2 fluorescence measurements, forskolin-stimulated Cl^– secretion occurred in the absence of changes in intracellular Ca²⁺. Thus, we conclude that there is an apical Cl^– channel in HT-29/B6 that is activated through the cAMP-mediated pathway and is insensitive to NPPB and DNDS, and the kinetics of which are incompatible with ORDIC kinetics. Therefore, despite its prevalence in isolated patches and even in cell-attached recordings, the ORDIC appears not to be involved in cAMP-mediated Cl^– secretion by HT-29/B6 cells. From noise analysis, a very-small-conductance (probably below 1 pS), slow-gating Cl^– channel was calculated as the conductive site in the apical membrane during forskolin stimulation.     

Arachidonic acid activation of BKCa (Slo1) channels associated to the β1-subunit in human vascular smooth muscle cells

Arachidonic acid (AA) is a polyunsaturated fatty acid involved in a complex network of cell signaling. It is well known that this fatty acid can directly modulate several cellular target structures, among them, ion channels. We explored the effects of AA on high conductance Ca²⁺- and voltage-dependent K⁺ channel (BK_Ca) in vascular smooth muscle cells (VSMCs) where the presence of β₁-subunit was functionally demonstrated by lithocholic acid activation. Using patch-clamp technique, we show at the single channel level that 10 μM AA increases the open probability (Po) of BK_Ca channels tenfold, mainly by a reduction of closed dwell times. AA also induces a left-shift in Po versus voltage curves without modifying their steepness. Furthermore, AA accelerates the kinetics of the voltage channel activation by a fourfold reduction in latencies to first channel opening. When AA was tested on BK_Ca channel expressed in HEK cells with or without the β₁-subunit, activation only occurs in presence of the modulatory subunit. These results contribute to highlight the molecular mechanism of AA-dependent BK_Ca activation. We conclude that AA itself selectively activates the β₁-associated BK_Ca channel, destabilizing its closed state probably by interacting with the β₁-subunit, without modifying the channel voltage sensitivity. Since BK_Ca channels physiologically contribute to regulation of VSMCs contractility and blood pressure, we used the whole-cell configuration to show that AA is able to activate these channels, inducing significant cell hyperpolarization that can lead to VSMCs relaxation.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

Small-conductance Cl− channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP

The present study demonstrates the activation of Cl^– channels in HT₂₉ cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca²⁺, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast wholecell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with halfmaximal effects at 0.4 umol/l for ATP, 60 pmol/l for neurotensin and 0.8 mol/l for carbachol. The depolarization, which was caused by Cl^– conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295±32 pS to 1180±271 pS (ATP; 10 mol/l, n=21) and 192±37 pS to 443±128 pS (neurotensin; 1 nmol/l, n=8). The effects of the agonists could be mimicked by ionomycin (0.2 umol/l), suggesting that an increase in intracellular Ca²⁺ was responsible for the activation of Cl^– channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10±0.8 mV and the cell-attached conductance increased from 228±29 pS to 410±65 (n=26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments.5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) did not prevent the effects of Ca²⁺-increasing hormones and of hypotonic solutions. An increase in Cl^– conductance was also induced by 8-Br-cGMP (1 mmol/l) but not by heat-stable Escherichia coli toxin. In contrast to their conductance-increasing effects in CAN patches, the different agonists and cell swelling did not activate resolvable single channels in these cell-attached membranes. This indicates that the Cl^– channels involved have a single-channel conductance too small ( 4 pS, 150 Hz) to be resolved by our techniques.     

Dual regulation of M current in gastric smooth muscle cells: β-adrenergic-muscarinic antagonism

The effects of the -adrenergic agent isoproterenol on membrane currents were studied in freshly dissociated gastric smooth muscle cells of Bufo marinus. Voltage-clamp experiments were carried out with patch pipettes in the tight-seal, whole-cell recording mode or with conventional microelectrodes. Isoproterenol induced a current identified as M current by the following criteria: the induced current is outward and carried by K⁺ ions, is suppressed by muscarine or acetylcholine, remains steadily activated, turns off with hyperpolarization, and exhibits slow relaxations in response to voltage jumps. In contrast to endogenous M current, isoproterenol-induced M current usually exhibited slower relaxations on hyperpolarizing voltage commands and displayed a steady-state conductance/voltage relationship that was shifted in the negative direction along the voltage axis. M current was also induced by either forskolin or phosphodiesterase-resistant cAMP analogs. In all cases, muscarinic agonists suppressed the M current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. -Adrenergic agents may act to increase the number of M channels available to be opened and also modify their kinetics.     

Influence of extracellur Ca2+ on endogenous Cl− channels in Xenopus oocytes

Removal of Ca²⁺ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl^– efflux, which could be strongly inhibited by the Cl^– channel blockers flufenamic acid and niflumic acid. Removal of Mg²⁺ or Ba²⁺ from the solutions had the same effects as removing Ca²⁺. The reversal potential of –12 mV also indicated that Cl^– channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca²⁺ concentration of about 20 M showed that quite low doses of extracellular Ca²⁺ profoundly influence the electrical properties of the oocyte membrane.     

N-Acetyl-l-cysteine and its derivatives activate a Cl− conductance in epithelial cells

N-Acetyl-l-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (we) conductance (G _m) and voltage (V _m) of NAC and the congeners S-carboxymethyl-l-cysteine (CMC) andS-carbamyl-L-cysteine (CAC) andL-cysteine in normal and CF airway epithelial cells.L-Cysteine (1 mmol/1) had no detectable effect. The increase inG _m (G_m) by the other compounds was concentration dependent and was (all substances at 1 mmol/1) 3.8 ± 1.4 nS (NAC; n = 11), 4.2 ± 1.0 nS (CMC;n = 16) and 3.8 ± 1.6 nS (CAC;n = 18), respectively. The changes in G_m were paralleled by an increased depolarization (V_m) when extracellular Cl^– concentration was reduced to 34 mmol/1: under control conditions = -4.1 ± 2.1 versus 10.2 ± 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl^– concentration was paralleled by a reduction ofG _m by 2.1 ± 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl^– conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/1). A Cl^– conductance was also activated in HT₂₉ colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPA1 cells, which do not express detectable levels of F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl^– currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-l-methylxanthine (IBMX) (Gm = 0.08 ±0.04 S;n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected cocytesG _m was enhanced when intracellular adenosine 3,5-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (G _m = 4.5 ± 1.3 S;n = 8).G _m was significantly increased by 0.74 ± 0.2 S (n = 11) and 0.46 ± 0.1 S (n = 10) when oocytes were exposed to NAC and CMC, respectively (both I mmol/1). In conclusion, NAC and its congeners activate Cl^– conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.     

Airway smooth muscle: immunomodulatory cells that modulate airway remodeling?

Although the pathogenesis of asthma remains unclear, substantial progress has been made over the past decades in the characterization of airway inflammation as a pathogenetic mechanism in asthma. New evidence suggests that airway smooth muscle (ASM), the most important cell modulating bronchomotor tone, plays an important immunomodulatory role in the orchestration and perpetuation of airway inflammation. Evidence now suggests that the signaling pathways that modulate leukocyte function may be disparate from those found in resident effector cells such as ASM, fibroblasts and epithelial cells. Further investigation and understanding of the critical signaling pathways that modulate ASM cell release, secretion of chemokines/cytokines and expression of cell adhesion molecules (CAMs) may offer new therapeutic approaches in the treatment of asthma.