Tracheal Smooth Muscle Cells Research Articles

Intracellular calcium stores in isolated tracheal smooth muscle cells.

We investigated the effects of acetylcholine (ACh) and histamine on intracellular calcium concentration ([Ca2+]i) and contraction of freshly isolated guinea pig tracheal smooth muscle cells. Previous electrophysiological studies revealed that agonists elicit cation and Cl- currents, but a role for Ca2+ in mediating these effects remains unresolved. Here we characterize agonist-induced changes of [Ca2+]i, using fura 2, and examine the contribution of the sarcoplasmic reticulum (SR) to regulation of [Ca2+]i. We provide evidence that the rise of [Ca2+]i and the contraction elicited by ACh or histamine are largely due to release of Ca2+ from stores. Agonists elicited Ca2+ transients in Ca(2+)-free solution with 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N, N, N',N'-tetraacetic acid (EGTA), whereas prolonged exposure to Ca(2+)-free solution diminished the rise of [Ca2+]i. In addition, blockade of SR Ca(2+)-adenosinetriphosphatase (ATPase) by cyclopiazonic acid (CPA) or thapsigargin caused elevation of [Ca2+]i and reduction of ACh-evoked increase of [Ca2+]i. In many cells, [Ca2+]i fell below baseline (undershoot) after ACh or caffeine. CPA abolished this undershoot and reduced the rate of recovery of [Ca2+]i to basal levels. Furthermore, oscillations of [Ca2+]i were elicited in the presence or absence of extracellular Ca2+, and these too were reversibly abolished by CPA. Our results provide evidence that Ca2+ stores play a significant role in agonist-mediated increase of [Ca2+]i in tracheal muscle and that the SR contributes to the restoration of basal Ca2+ levels.

Regulation of 5-hydroxytryptamine-induced calcium mobilization by cAMP-elevating agents in cultured canine tracheal smooth muscle cells.

The effects of increases in cellular adenosine 3'5'-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca2+ ([Ca2+]i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC50) produced at concentrations of 7.0 +/- 0.5 and 4.9 +/- 0.4 respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca2+ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF4--induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF4-- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca2+]i increase or inhibit both responses independently.

Activation of the TNF alpha-p55 receptor induces myocyte proliferation and modulates agonist-evoked calcium transients in cultured human tracheal smooth muscle cells.

Evidence suggests that cytokines may modulate smooth muscle cell function in a variety of inflammatory diseases. In the present study, we characterized the specific receptor subtypes that mediate tumor necrosis factor alpha (TNF alpha) effects on myocyte proliferation and on agonist-induced calcium transients in cultured human tracheal smooth muscle cells (TSMC). Pretreatment of human TSMC with TNF alpha potentiated cytosolic calcium [(Ca2+)i] transients evoked by carbachol. In a similar manner, selective TNF alpha-p55 receptor agonists such as htr-9, an activating monoclonal antibody, or a recombinant TNF-p55 (rTNF-p55), which specifically activates the TNF alpha-p55 receptor but not the TNF alpha-p75 receptor, also augmented [Ca2+]i transients evoked by carbachol. In parallel experiments, TNF alpha, rTNF alpha-p55, and htr-9 induced human TSMC proliferation as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Interestingly, activation of the TNF alpha-p75 receptor with a selective agonist, recombinant TNF alpha-p75 (rTNF alpha-p75), or inhibition of the TNF alpha-p75 receptor with utr-1, an inhibitory anti-TNF alpha-p75 receptor antibody, had no effect on TNF alpha-augmented calcium transients or on myocyte growth. To further confirm the receptor specificity of these findings, immunocytochemical studies were performed using receptor-specific antibodies. These studies demonstrated marked cell-surface expression of the TNF alpha-p55 receptor compared with expression of the TNF alpha-p75 receptor on human TSMC. Taken together, our results suggest that TNF alpha modulates agonist-induced calcium transients and induces human TSMC proliferation by specific activation of the TNF alpha-p55 receptor. Further studies addressing the cellular and molecular mechanisms regulating cytokine modulation of airway smooth muscle function may provide new insight into mechanisms that induce airway hyperresponsiveness in asthma.

Identification of Promoter Elements Involved in Cell-specific Regulation of Rat Smooth Muscle Myosin Heavy Chain Gene Transcription

In order to identify cis-acting regulatory elements involved in smooth muscle cell-specific gene regulation, we have cloned a 4. 7-kilobase pair fragment of the promoter for the rat smooth muscle myosin heavy chain, a protein expressed in differentiated smooth muscle cells. Sequence analysis of a 1.7-kilobase pair portion of this clone reveals potential binding sites for known transcription factors. A comparison of the primary sequence between the rat and rabbit smooth muscle myosin heavy chain promoters reveals numerous conserved consensus binding sites. Transient transfection analysis of promoter deletion constructs in rat aorta and tracheal smooth muscle cells, L8 myoblast cells, and rat pulmonary aorta endothelial cells suggests that a region of the promoter located between -1,249 and -1,317 base pairs is important for the restriction of gene expression to smooth muscle cells. Electrophoretic mobility shift analysis of a highly conserved region located between -1,317 and -1, 085 base pairs reveals specific DNA-protein complexes formed in smooth muscle cell extracts, which can be competed with an oligonucleotide containing a nuclear factor 1 binding site.

Inhibition by endothelin-1 of cholinergic nerve-mediated acetylcholine release and contraction in sheep isolated trachea.

1. The relative roles of ETA and ETB receptor activation on cholinergic nerve-mediated contraction and acetylcholine (ACh) release were examined in sheep isolated tracheal smooth muscle. 2. Electrical field stimulation (EFS; 90 V, 0.5 ms duration, 1 Hz, 10 s train) applied to sheep isolated tracheal smooth muscle strips induced monophasic contractile responses that were abolished by either 1 microM tetrodotoxin or 0.1 microM atropine, but were insensitive to 10 microM hexamethonium and 100 microM L-NAME. Thus, EFS-induced contractions resulted from the spasmogenic actions of ACh released from parasympathetic, postganglionic nerves. 3. As expected, sheep isolated tracheal smooth muscle preparations did not contract in response to the ETB receptor-selective agonist, sarafotoxin S6c (0.1-100 nM). However, sarafotoxin S6c caused a concentration-dependent and transient inhibition of EFS-induced contractions. The inhibitory effect induced by a maximally effective concentration of sarafotoxin S6c (10 nM; 72.1 +/- 5.7%, n = 6) was abolished in the presence of the ETB receptor-selective antagonist BQ-788 (1 microM). Contractile responses to exogenously administered ACh (10 nM-0.3 mM) were not inhibited by sarafotoxin S6c (1 or 10 nM; n = 7). 4. In contrast to sarafotoxin S6c, endothelin-1 induced marked contractions in sheep isolated tracheal smooth muscle. These contractions were inhibited by BQ-123, consistent with an ETA receptor-mediated response. In the presence of BQ-123 (3 microM), endothelin-1 produced a concentration-dependent inhibition of EFS-induced contractions (30 nM endothelin-1, 68.9 +/- 10.2% inhibition, n = 5). These responses were inhibited by 1 microM BQ-788, indicative of an ETB receptor-mediated process. Endothelin-1 was about 3 fold less potent than sarafotoxin S6c. 5. EFS (90 V, 0.5 ms duration, 1 Hz, 15 min train) induced the release of endogenous ACh (1.94 +/- 0.28 pmol mg-1 tissue, n = 12), as assayed by h.p.l.c. with electrochemical detection. EFS-induced release of ACh was inhibited to a similar extent by 100 nM endothelin-1 (47 +/- 4%, n = 9) and 10 nM sarafotoxin S6c (46 +/- 9%, n = 3). These effects of endothelin-1 on ACh release were inhibited by 1 microM BQ-788 alone (n = 4), by BQ-788 in the presence of 3 microM BQ-123 (n = 4), but not by 3 microM BQ-123 alone (n = 5). 6. In summary, sheep isolated tracheal smooth muscle contains two anatomically and functionally distinct endothelin receptor populations. ETA receptors located on airway smooth muscle mediate contraction, whereas ETB receptors appear to exist on cholinergic nerves that innervate tracheal smooth muscle cells and mediate inhibition of ACh release. The inhibitory effect of ETB receptor stimulation on cholinergic neurotransmission is in stark contrast to the enhancing effects hitherto described in the airways.

Markers of airway smooth muscle cell phenotype.

Airway smooth muscle plays a principal role in the pathogenesis of asthma. Primary cultures are being used to investigate airway myocyte proliferation and cellular pathways regulating contraction. Airway smooth muscle cells (SMC) modulate from a contractile to a noncontractile phenotype in culture, but no systematic study of the concomitant changes in expression of cytocontractile and cytoskeletal proteins has been reported. We measured temporal changes in protein marker expression of canine tracheal SMC in primary culture, using specific antibodies and cDNA probes. Immunoblot analysis revealed that when cells became proliferative after 5 days of culture, the content of smooth muscle myosin heavy chain (sm-MHC), calponin, sm-alpha-actin, and desmin diminished by > 75%; myosin light chain kinase, h-caldesmon, and beta-tropomyosin had also decreased significantly (P < 0.05). Northern blots revealed that mRNA levels for sm-MHC and sm-alpha-actin were also significantly reduced in proliferative SMC. Conversely, immunoblotting demonstrated the content of non-muscle myosin heavy chain, l-caldesmon, vimentin, alpha/beta-protein kinase C (PKC), and CD44 homing cellular adhesion molecule (HCAM) increased one- to sixfold as cells became proliferative. The content of sm-MHC and sm-alpha-actin protein increased after confluence, suggesting that cultured airway SMC are capable of phenotypic plasticity. Marker protein contents were also compared, by immunoblot assay, between SMC dissociated from trachealis or pulmonary arterial media. Cytocontractile protein content was higher in the trachea, which shortens faster than the pulmonary artery. The identification of these markers provides tools for assessing the phenotype of airway SMC in culture and the airways of asthmatic patients.

Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon.

1. In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin-1 (ET-1), ET-3, and sarafotoxin S6c (S6c), the ETB receptor-selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2. In confluent, growth-arrested human airway smooth, neither ET-1 (0.01 nM-1 microM) nor ET-3 (0.001 nM-1 microM) or S6c (0.01 nM-1 microM) induced cell proliferation, as assessed by [3H]-thymidine incorporation. In contrast, EGF (1.6 pM-16 nM) produced concentration-dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16 nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16-16 nM) also produced a concentration-dependent increase in cell counts, whereas ET-1 (1-100 nM) was without effect on this index of mitogenesis. 3. ET-1 (1-100 nM) potentiated EGF-induced proliferation of human tracheal smooth muscle cells. For example, ET-1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET-1 on EGF-induced proliferation was antagonized by BQ-123 (3 microM), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ-788 (10 microM). 4. Neither ET-3 (1-100 nM) nor S6c (1-100 nM) influenced the mitogenic effects of EGF (0.16-1.6 nM). 5. [125I]-ET-1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 ( +/- 3; n = 4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6. These data indicate that ET-1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor-mediated mechanism. These findings suggest that ET-1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.

EFFECT OF SUPEROXIDE DISMUTASE INHIBITOR ON NITRIC OXIDE-INDUCED AIRWAY RELAXATION AND INTRACELLULAR Ca2+ TRANSIENTS. ▴ 2307

Endogenous production of superoxide anion (SA) is known to affect a variety of signal transduction pathways in smooth muscle. In this study, we investigated the effect of diethyldithiocarbamate (DETCA), an inhibitor of Zn, Cu-superoxide dismutase (SOD), on nitric oxide (NO)-mediated relaxation and intracellular Ca2- ([Ca2+]i) transients in porcine tracheal smooth muscle (TSM) cells. In carbachol (CH)-contracted TSM strips, DETCA (10 mM) inhibited relaxations mediated by cGMP and induced by NO released from intrinsic nerves as well as by the NO donor, sodium nitroprusside (SNP), but not that mediated by cAMP and induced by isoproterenol. In CH-contracted tissues, exposure to DETCA resulted in increased lucigenin-dependent chemiluminescence. DETCA did not inhibit the elevation of cGMP in response to SNP. In single, freshly isolated TSM cells loaded with fura-2/AM, exposure to 100 nM acetylcholine (ACh), which acts through the inositol 1,4,5-trisphosphate (IP3) receptor, resulted in elevation of [Ca2+]i. DETCA attenuated this Ca2+ response, but not to caffeine, an inducer of Ca2+ release through the ryanodine receptor. These results suggest that SA attenuates NO-mediated relaxations at a step distal to the generation of cGMP, and decreases ACh-induced elevation of [Ca2+]i by inhibiting IP3-induced Ca2+ release selectively. Supported by Univ. of Minnesota, Mayo Foundation, and NIH grant HL51736-02C. MSK was recipient of an Abbott Senior Fellowship.

MODULATION OF INTRACELLULAR Ca2+ TRANSIENTS BY NITRIC OXIDE IN AIRWAY SMOOTH MUSCLE CELLS. ▴ 2308

Nitric oxide (NO) causes tracheal smooth muscle (TSM) relaxation by inhibiting agonist-stimulated elevation of intracellular Ca2+([Ca2+]i). via cGMP-dependent mechanisms. In this study, we examined the effect of s-nitroso-n-acetylpenicillamine (SNAP), a NO donor, on the Ca2+ response to acetylcholine (ACh). Single, freshly isolated porcine TSM cells were loaded with fura-2/AM and [Ca2+]i was measured as the ratio of fura-2 emissions when excited at 340 and 380 nm, using a video fluorescence imaging system. SNAP (0.1-1.0 μM) inhibited the normal [Ca2+]i response to ACh (0.1-1.0 μM) at an extracellular Ca2+ ([Ca2+]o) of either 0 or 2.5 mM. After SNAP inhibition of [Ca2+]i response to ACh in 0 mM Ca2+, a large Ca2+ response was seen when [Ca2+]o was readjusted to 2.5 mM, indicating that Ca2+ influx was not affected by NO. After depleting the sarcoplasmic reticulum (SR) Ca2+ pool with ACh and re-exposing to 2.5 mM Ca2+ in the presence of SNAP, the subsequent ACh response in the absence of SNAP was unaffected, indicating that NO does not affect reloading of the SR. These results suggest that NO selectively affects SR Ca2+ release, but does not affect Ca2+ influx or SR reloading. Inhibition of SR Ca2+ release can lead to relaxation of TSM. Supported by NIH grants HL51736 and HL07111, Mayo Foundation, and Univ. of Minnesota. MSK was a recipient of an Abbott Senior Fellowship.

Effects of CAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

The effects of increases in intracellular adenosine 3′,5′-cyclic monophosphate (CAMP) on carbachol-induced generation of inositol phosphates (IPs) and increases in intracellular Ca 2+ ([Ca 2+] i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The CAMP elevating agents, cholera toxin (CTX) and forskolin, induced concentration- and time-dependent CAMP formation with half-maximal effects (-IogEC 50) at concentrations of 7.6 ± 1.3 g/ml and 4.8 ± 0.9 M, respectively. Forskolin caused a concentration-dependent inhibition of carbachol-induced increase in [Ca 2+] i with half-maximal inhibition (-IogEC 50 at 5.2 ± 0.7 M. Pretreatment of TSMCs with either CTX (10 μ/ml, 4 h), forskolin (10–100 μM, 30 min), or dibutyryl CAMP (1 mM, 30 min) inhibited carbachol-stimulated Ca 2+ mobilization and IPs accumulation. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of carbachol without changing the EC 50 values. After treatment with forskolin for 24 h, carbachol-induced IPs accumulation and Ca 2+ mobilization were close to those of control group. SQ-22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 μM], an inhibitor of adenylate cyclase, and HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, 50 μM], an inhibitor of CAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit carbachol-induced IPs accumulation. Moreover, the inactive analogue of forskolin, 1,9-dideoxy forskolin, did not inhibit these responses evoked by carbachol, suggesting that activation of CAMP/PKA was involved in these inhibitory effects of forskolin. The K D and B max values of the muscarinic receptor (mAChR) for [ 3H]-N-methyl scopolamine binding were not significantly changed by forskolin treatment for 30 min and 24 h, suggesting that the inhibitory effect of forskolin is distal to the mAChR. The locus of this inhibition was further investigated by examining the effect of forskolin treatment on AIF 4-stimulated IPs accumulation in canine TSMCs. The AIF 4-induced response was inhibited by forskolin, supporting the notion that G protein(s) are directly activated by AIF4 and uncoupled to phospholipase C by forskolin treatment. We conclude that CAMP elevating agents inhibit carbachol-stimulated generation of IPs and Ca 2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca 2+]; are very early events in the activation of mAChRs, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of CAMP on tracheal smooth muscle function.

Cholinergic inhibition of Ca2+ current in guinea‐pig gastric and tracheal smooth muscle cells.

1. Cholinergic regulation of L-type Ca2+ channels was investigated in freshly dissociated guinea-pig gastric and tracheal smooth muscle cells. Acetylcholine (ACh, 50 microM) decreased Ca2+ channel current (ICa) by 37 +/- 3% (mean +/- S.E.M., 46 cells). 2. ACh reduced ICa at all voltages, with no shift in the current-voltage relationship. Effects of ACh were rapid (within 5 s) and repeatable, with multiple applications reproducibly inhibiting ICa in the continued presence of extracellular Ca2+ and in the presence of protein kinase C inhibitors. 3. The involvement of Ca2+ stores in this inhibition was investigated using Ca(2+)-free solution or cyclopiazonic acid (CPA) to deplete the stores. ACh initially inhibited ICa in the Ca(2+)-free solution (Na+ as charge carrier, 53 +/- 4% decrease, 18 cells) with subsequent responses significantly attenuated (n = 9). CPA (1 microM) reduced, then abolished, the effects of ACh on ICa (n = 5). 4. When studied in cell-attached patches (Ba2+ as charge carrier), ACh reduced Ca2+ channel open probability in twenty-two of thirty-six cells, consistent with the involvement of a diffusible cytosolic messenger. 5. ACh also inhibited ICa in tracheal muscle cells (reduction of 38 +/- 6% in 1 mM Ca2+, 4 cells; 77 +/- 3% in Ca(2+)-free solution, 7 cells). Furthermore, in cells where ACh elicited oscillating Ca(2+)-activated Cl- current, oscillatory inhibition of ICa was also observed (3 cells). 6. In summary, ACh causes rapid and reversible inhibition of ICa in gastric and tracheal muscles. Ca2+ stores were required to initiate this effect, with the rapid onset and oscillatory inhibition consistent with Ca2+ inhibition of the channel. Suppression of ICa would reduce Ca2+ entry during cholinergic excitation.

Sodium nitroprusside stimulates Ca2+ -activated K+ channels in porcine tracheal smooth muscle cells.

To directly investigate the possible role of large-conductance Ca2+ -activated K+ (KCa) channels in nitro-vasodilator-induced relaxation of airway smooth muscle, we used cell-attached patch-clamp techniques to test the effects of sodium nitroprusside (SNP) on KCa channels in freshly dispersed porcine tracheal smooth muscle cells. Channel open-state probability (nPo) increased approximately 13-fold with exposure to 10(-5) M SNP, and this was partially reversed by addition of the guanylate cyclase inhibitors methylene blue (3 X 10(-4) M) or LY-83583 (5 X 10(-5) M). Pretreatment with the guanosine 3',5' -cyclic monophosphate (cGMP)-dependent protein kinase (G kinase) inhibitor Rp-8-(p-chlorophenylthio) cGMP-phosphorothioate (2 X 10(-5) M) prevented activation of KCa channels by SNP. We also tested the ability of G kinase to directly activate KCa channels in inside-out patches. G kinase (2.5 U/microliter) with ATP (0.5 mM) and cGMP (0.1 mM), but not ATP and cGMP alone, increased nPo approximately 23-fold. We conclude that SNP activates KCa channels in airway smooth muscle via guanylate cyclase and G kinase. Phosphorylation of the channel protein by G kinase may account for this response. Consequent membrane hyperpolarization and inhibition of Ca2+ entry through voltage-dependent channels may contribute to SNP-induced relaxation of airway smooth muscle.

Modulation of agonist-induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms.

1. The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells. 2. Isoprenaline (pD2 (-log10 EC50) = 6.32 +/- 0.24) and forskolin (pD2 = 5.6 +/- 0.05) enhanced cyclic AMP levels in a concentration-dependent fashion in these cells, while methacholine (pD2 = 5.64 +/- 0.12) and histamine (pD2 = 4.90 +/- 0.04) caused a concentration-related increase in [3H]-inositol phosphates (IP) accumulation in the presence of 10 mM LiCl. 3. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0 +/- 0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 microM. 4. Both methacholine and histamine induced a fast (max. in 0.5-2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+. 5. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 microM) significantly attenuated both the Ca(2+)-transient and the sustained phase generated at equipotent IP producing concentrations of 1 microM methacholine and 100 microM histamine (approx. 40% of maximal methacholine-induced IP response), but did not affect changes in [Ca2+]i induced by 100 microM methacholine (95.2 +/- 3.5% of maximal methacholine-induced IP response). 6. Significant correlations were found between the isoprenaline-induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist. 7. These data indicate that (a) cyclic AMP-dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+]i homeostasis, (b) that attenuation of intracellular Ca2+ concentration plays a major role in beta-adrenoceptor-mediated relaxation of methacholine- and histamine-induced airway smooth muscle contraction, and (c) that the relative resistance of the muscarinic agonist-induced contraction to beta-adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined by its higher potency in inducing intracellular Ca2+ changes.

Pharmacological Characteristics of BDTI, a New Isoquinoline-Derived &amp;beta;&lt;sub&gt;2&lt;/sub&gt;-Adrenoceptor Agonist, in Canine Trachea and Rat Heart

The tracheal relaxing effects and beta 2-selectivity of BDTI (1-benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline HBr) were investigated in canine trachea and rat heart by radioligand binding assay and pharmacological experiments in comparison with those of other beta-adrenoceptor agonists, salbutamol and isoprenaline. The potency of relaxing effect on carbachol-induced contraction in isolated canine trachea was in the order of isoprenaline (pD2 = 6.70 +/- 0.08) > BDTI (6.11 +/- 0.06) approximately salbutamol (6.14 +/- 0.08). ICI-118,551 (a selective beta 2-antagonist) and atenolol (a selective beta 1-antagonist) inhibited the relaxant action of BDTI with pKB values of 8.4 and 5.3, respectively, corresponding to high affinity for ICI-118,551 and low affinity for atenolol in antagonizing this response. The Kd values of radioligand ([3H]-CGP12177) were 453.3 +/- 30.8 and 563.4 +/- 96.7 pmol/l in cultured canine tracheal smooth muscle cells (TSMCs) and rat cardiomyocytes, respectively, and the Bmax values were 64.6 +/- 10.7 and 245.7 +/- 44.5 fmol/mg protein, respectively. BDTI, salbutamol and isoprenaline inhibited the binding of [3H]-CGP12177 in a concentration-dependent manner in cultured canine TSMCs (Ki 0.73 +/- 0.15, 0.75 +/- 0.21 and 0.24 +/- 0.05 mumol/l, respectively) and rat cardiomyocytes (Ki 2.76 +/- 0.36, 2.31 +/- 0.26 and 0.22 +/- 0.03 mumol/l, respectively). These results demonstrated that BDTI possessed moderate selectivity (3.8-fold) to beta 2-adrenoceptors as judged from the Ki (heart)/Ki (trachea) value (salbutamol 3.1-fold, isoprenaline 0.92-fold). BDTI and salbutamol also stimulated cAMP formation in a concentration-dependent manner in cultured canine TSMCs (EC50 0.5 +/- 0.2 and 0.4 +/- 0.1 mumol/l, respectively) and rat cardiomyocytes (EC50 6.2 +/- 0.5 and 5.7 +/- 0.6 mumol/l, respectively). The selectivity of BDTI and salbutamol for beta 2-adrenoceptors on the cAMP response were 12.4 and 14.3 times, respectively. It is concluded that BDTI is a beta 2-selective adrenoceptor agonist.

P-229 - The role of endothelins in the porcine tracheal smooth muscle and epithelial cells.

P-229 - The role of endothelins in the porcine tracheal smooth muscle and epithelial cells.

P-611 - The activation of Ca2+-dependent Cl- current and changes in intracellular Ca2+ concentration in tracheal smooth muscle cells of the guinea-pig.

P-611 - The activation of Ca2+-dependent Cl- current and changes in intracellular Ca2+ concentration in tracheal smooth muscle cells of the guinea-pig.

Cholinergic regulation of voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells.

To investigate cholinergic regulation of voltage-dependent Ca2+ channels (VDCs) in airway smooth muscle, we measured inward currents through VDCs in enzymatically dispersed porcine tracheal smooth muscle cells using conventional (10 mM Ca2+ as charge carrier) and nystatin-perforated (5 mM Ba2+ as charge carrier) whole cell patch clamp techniques. Carbachol (CCh) had significant and dose-dependent inhibitory effects on inward currents (12% with 10(-7) M and 42% with 10(-6) M) in perforated whole cell clamp experiments, but had no effect on currents in conventional whole cell experiments. CCh also shifted the steady-state inactivation curve to more negative potentials. Further experiments tested the hypothesis that CCh inhibits VDCs in part by the activation of protein kinase C (PKC). Phorbol 12,13-diacetate, an exogenous PKC activator, inhibited currents through VDCs. and calphostin C, a specific PKC inhibitor, antagonized the inhibitory effect of CCh. Furthermore, intracellular exposure to the activating PKC fragment 530-558, using a pipette perfusion technique, also inhibited currents through VDCs. We conclude that cholinergic receptor stimulation can inhibit inward Ca2+ currents through VDCs of porcine tracheal smooth muscle and that this effect may be mediated in part by activation of PKC.

Arachidonic acid metabolites and glucocorticoid regulatory mechanism in cultured porcine tracheal smooth muscle cells.

To elucidate the signal transduction system in the production of prostaglandin E2 (PGE2) by porcine tracheal smooth muscle cells in culture (PTSMC), we examined the pattern of arachidonic acid metabolites released from PTSMC and the relationship between bradykinin-stimulated rises in intracellular calcium concentration ([Ca2+]i) and PGE2 production by PTSMC. We next examined the effect of dexamethasone on these parameters. Bradykinin induced a dose-dependent increase in both the rise in [Ca2+]i and PGE2 production by PTSMC. The increase in [Ca2+]i paralleled an increase in PGE2 production. High-performance liquid chromatography (HPLC) revealed that dexamethasone-treated PTSMC were suppressed to release arachidonic acid metabolites such as PGE2 and prostaglandin F2 alpha (PGF2 alpha). Incubation of PTSMC with 10(-6)M dexamethasone for 24 h significantly suppressed both the rise in [Ca2+]i and PGE2 production by PTSMC in response to bradykinin, and also significantly suppressed bradykinin-stimulated release of radioactivity from PTSMC prelabeled with 3H-labeled arachidonic acid (3H-AA). When PTSMC pretreated with dexamethasone were incubated with 170 nM prostaglandin H2 (PGH2) or 20 microM arachidonic acid; PTSMC synthesized less PGE2 than control PTSMC. Results suggest that bradykinin stimulates PTSMC to produce PGE2 via the signal transduction system including Ca2+, and dexamethasone appeared to suppress PGE2 production by reducing the activity of cytosolic phospholipase A2 (cPLA2) and PGE2 synthase. However, we failed to demonstrate the suppression of the activity of cyclooxygenase in PTSMC by dexamethasone. Since the elevation of [Ca2+]i is necessary for the contraction of airway smooth muscles, dexamethasone seems to reduce the contraction of airway smooth muscles by suppressing the rise in [Ca2+]i and the release of arachidonic acid metabolites. Reduced production of arachidonic acid metabolites may also contribute to improvement in the bronchial inflammation.

Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.

1. In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2. Both ET-1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient. 3. Activation of MAP kinase occurred in a concentration-dependent manner (EC50 values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml-1). 4. Pretreatment with the protein kinase C (PKC) inhibitor Ro-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min. 5. Following chronic phorbol ester pretreatment, BK-stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET-1 were only partly reduced (80 and 45% inhibition respectively). 6. Pretreatment with pertussis toxin reduced ET-1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7. ET-1 also stimulated a 3 fold increase in [3H]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8. These results suggest that a pertussis toxin-sensitive activation of MAP kinase may play an important role in ET-1-stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF.

Ca2+-regulated Dynamic Compartmentalization of Calmodulin in Living Smooth Muscle Cells

A key assumption of most models for calmodulin regulation of smooth and non-muscle contractility is that calmodulin is freely diffusible at resting intracellular concentrations of free Ca2+. However, fluorescence recovery after photobleaching (FRAP) measurements of three different fluorescent analogs of calmodulin in cultured bovine tracheal smooth muscle cells suggest that free calmodulin may be limiting in unstimulated cells. Thirty-seven % of microinjected calmodulin is immobile by FRAP and the fastest recovering component has an effective diffusion coefficient 7-fold slower than a dextran of equivalent size. Combining the FRAP data with extraction data reported in a previous paper (Tansey, M., Luby-Phelps, K., Kamm, K.E., and Stull, J.T. (1994) J. Biol. Chem. 269, 9912-9920), we estimate that at most 5% of total endogenous calmodulin in resting smooth muscle cells is unbound (freely diffusible). Examination of the Ca2+ dependence of calmodulin mobility in permeabilized cells reveals that binding persists even at intracellular Ca2+ concentrations as low as 17 nM. When Ca2+ is elevated to between 450 nM and 3 microM, some of the bound calmodulin is released, as indicated by an increase in the effective diffusion coefficient and the percent mobile fraction. At higher Ca2+, calmodulin becomes increasingly immobilized. In about 50% of the cell population, clamping Ca2+ at micromolar levels results in translocation of cytoplasmic calmodulin to the nucleus. The compartmentalization and complex dynamics of calmodulin in living smooth muscle cells have profound implications for understanding how calmodulin regulates contractility in response to extracellular signals.