Canine Tracheal Smooth Muscle Cells Research Articles

Nuclear Import of Serum Response Factor in Airway Smooth Muscle

We have previously shown that the transcription-promoting activity of serum response factor (SRF) is partially regulated by its extranuclear redistribution. In this study, we examined the cellular mechanisms that facilitate SRF nuclear entry in canine tracheal smooth muscle cells. We used in vitro pull-down assays to determine which karyopherin proteins bound SRF and found that SRF binds KPNA1 and KPNB1 through its nuclear localization sequence. Immunoprecipitation studies also demonstrated direct SRF-KPNA1 interaction in HEK293 cells. Import assays demonstrated that KPNA1 and KPNB1 together were sufficient to mediate rapid nuclear import of SRF-GFP. Our studies also suggest that SRF is able to gain nuclear entry through an auxiliary, nuclear localization sequence-independent mechanism.

Involvement of MAPKs, NF-κB and p300 co-activator in IL-1β-induced cytosolic phospholipase A 2 expression in canine tracheal smooth muscle cells

Cytosolic phospholipase A 2 (cPLA 2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during stimulation with interleukin-1β (IL-1β). However, the mechanisms underlying IL-1β-induced cPLA 2 expression and PGE 2 synthesis by canine tracheal smooth muscle cells (CTSMCs) have not been defined. IL-1β induced cPLA 2 protein and mRNA expression, PGE 2 production, and phosphorylation of p42/p44 MAPK, p38 MAPK (ATF 2), and JNK (c-Jun) in a time- and concentration-dependent manner, determined by Western blotting, RT-PCR, and ELISA, which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), or transfection with dominant negative mutants of MEK1/2, p38, and JNK, respectively. Furthermore, IL-1β-induced cPLA 2 expression and PGE 2 synthesis was inhibited by a selective NF-κB inhibitor (helenalin) or transfection with dominant negative mutants of NF-κB inducing kinase (NIK), IκB kinase (IKK)-α, and IKK-β. Consistently, IL-1β stimulated both IκB-α degradation and NF-κB translocation into nucleus in these cells. NF-κB translocation was blocked by helenalin, but not by U0126, SB202190, and SP600125. MAPKs together with NF-κB-activated p300 recruited to cPLA 2 promoter thus facilitating the binding of NF-κB to cPLA 2 promoter region and expression of cPLA 2 mRNA. IL-1β-induced cPLA 2 expression and PGE 2 production was inhibited by actinomycin D and cycloheximide, indicating the involvement of transcriptional and translational events in these responses. These results suggest that in CTSMCs, IL-1β-induced cPLA 2 expression and PGE 2 synthesis was independently mediated through activation of MAPKs and NF-κB pathways and was connected to p300 recruitment and activation.

Involvement of MAPKs and NF‐κB in cPLA 2 induced by IL‐1β in tracheal smooth muscle cells

cPLA2 plays a pivotal role in mediating agonist-induced AA release for PG synthesis during stimulation with IL-1β. However, the signaling pathways mediating cPLA2 expression and PGE2 synthesis by canine tracheal smooth muscle cells (TSMCs) remain unknown. IL-1β-induced cPLA2 expression and PGE2 release were attenuated by inhibitors of tyrosine kinase (genistein), PC-PLC (D609), PI-PLC (U73122), PKC (GF109203X and staurosporine), Ca2+ (BAPTA+EDTA), MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and PI3-K (LY294002 and wortmannin). In addition, IL-1β-induced cPLA2 expression and PGE2 synthesis was attenuated by transfection with dominant negative mutants of NF-κB inducing kinase (NIK) and IκB kinase (IKK)-α, but not by IKK-β. Taken together, these findings suggest that the increased expression of cPLA2 correlates with the release of PGE2 from IL-1β-challenged TSMCs, at least in part, mediated through MAPKs and NF-κB signaling pathways. IL-1β-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in these cells.

Induction of cytosolic phospholipase A 2 by lipopolysaccharide in canine tracheal smooth muscle cells: Involvement of MAPKs and NF-κB pathways

Cytosolic phospholipase A 2 (cPLA 2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA 2 expression and PGE 2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA 2 and release of PGE 2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca 2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA 2 expression and PGE 2 synthesis was inhibited by a selective NF-κB inhibitor (helenalin) and transfection with dominant negative mutants of NF-κB inducing kinase (NIK), IκB kinase (IKK)-α, and IKK-β, consistent with that LPS-stimulated both IκB-α degradation and NF-κB translocation into nucleus in these cells. LPS-stimulated cPLA 2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA 2 and COX-2 linked to PGE 2 synthesis was inhibited by AACOCF 3 (a selective cPLA 2 inhibitor), implying the involvement of cPLA 2 in these responses. These findings suggest that phosphorylation and expression of cPLA 2 correlates with the release of PGE 2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-κB signaling pathways. LPS-mediated responses were modulated by PLC, Ca 2+, PKC, tyrosine kinase, and PI3-K in TSMCs.

Induction of cyclooxygenase-2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-κB pathways

Lipopolysaccharide (LPS) was found to induce inflammatory responses in the airways and exerted as a potent stimulus for PG synthesis. This study was to determine the mechanisms of LPS-enhanced cyclooxygenase (COX)-2 expression associated with PGE 2 synthesis in tracheal smooth muscle cells (TSMCs). LPS markedly increased the expression of COX-2 and release of PGE 2 in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Both the expression of COX-2 and the generation of PGE 2 in response to LPS were attenuated by a tyrosine kinase inhibitor genistein, a phosphatidylcholine-phospholipase C inhibitor D609, a phosphatidylinositol-phospholipase C inhibitor U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca 2+ by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. Furthermore, LPS-induced NF-κB activation correlated with the degradation of IκB-α, COX-2 expression, and PGE 2 synthesis, was inhibited by transfection with dominant negative mutants of NIK and IKK-α, but not by IKK-β. LPS-induced COX-2 expression and PGE 2 synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK inhibitor), but these two inhibitors had no effect on LPS-induced NF-κB activation, indicating that NF-κB is activated by LPS independently of activation of p42/p44 MAPK and p38 MAPK pathways in TSMCs. Taken together, these findings suggest that the increased expression of COX-2 correlates with the release of PGE 2 from LPS-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-κB signalling pathways. LPS-mediated responses were modulated by PLC, Ca 2+, PKC, tyrosine kinase, and PI3-K in these cells.

Interleukin-1β-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS, and NF-κB pathways in canine tracheal smooth muscle cells

Interleukin-β (IL-1β) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1β-enhanced cyclooxygenase (COX)-2 expression associated with PGE 2 synthesis in tracheal smooth muscle cells (TSMCs). IL-1β markedly increased COX-2 expression and PGE 2 formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE 2 formation in response to IL-1β were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca 2+ by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1β-induced activation of NF-κB correlated with the degradation of IκB-α in TSMCs. IL-1β-induced NF-κB activation, COX-2 expression, and PGE 2 synthesis were inhibited by the dominant negative mutants of NIK and IKK-α, but not by IKK-β. IL-1β-induced COX-2 expression and PGE 2 synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1β-induced NF-κB activation, indicating that activation of p42/44 and p38 MAPK and NF-κB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE 2 from IL-1β-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-κB signalling pathways in canine TSMCs. IL-1β-mediated responses were modulated by PLC, Ca 2+, PKC, tyrosine kinase, and PI3-K in these cells.

Tumour necrosis factor-α enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

Inhalation of tumour necrosis factor-α (TNF-α) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-α involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-α on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca 2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-α potentiated BK-induced IP accumulation and Ca 2+ mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-α and PDGF-BB might be due to an increase in BK B 2 receptor density ( B max), since [ 3H]BK binding to TSMCs was inhibited by the B 2 selective agonist and antagonist, BK and Hoe 140, but not by the B 1 selective reagents. The enhancing effects of TNF-α and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-α may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-α and PDGF-BB and attenuated the effect of TNF-α on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-α might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Interleukin-1beta enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth-muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway.

Elevated levels of several cytokines including interleukin-1beta (IL-1beta) have been detected in airway fluid of asthmatic patients. Inhalation of IL-1beta induced a bronchial hyper-reactivity to contractile agonists. However, the implication of IL-1beta in the pathogenesis of bronchial hyper-reactivity is not completely understood. Therefore, we investigated the effect of IL-1beta on bradykinin (BK)-induced inositol phosphate [Ins(X)P] accumulation and Ca2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth-muscle cells (TSMCs). Treatment of TSMCs with IL-1beta potentiated BK-induced Ins(X)P accumulation and Ca2+ mobilization. However, there was no effect on the Ins(X)P response induced by endothelin-1, 5-hydroxytryptamine or carbachol. Treatment with platelet-derived growth factor B-chain homodimer (PDGF-BB) also enhanced the BK-induced Ins(X)P response. These enhancements by IL-1beta and PDGF-BB might be due to an up-regulation of BK B(2) receptor density (B(max)), since [(3)H]BK binding to TSMCs was inhibited by the B(2)-selective agonist and antagonist, BK and Hoe 140, but not by B(1)-selective reagents. The enhancing effects of IL-1beta and PDGF-BB on Ins(X)P accumulation, Ca2+ mobilization and B(max) were attenuated by PD98059 [an inhibitor of activation of mitogen-activated protein kinase (MAPK) kinase, MEK] and cycloheximide (an inhibitor of protein synthesis), suggesting that IL-1beta may share a common signalling pathway with PDGF-BB via protein synthesis. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed the up-regulation of BK receptors induced by IL-1beta, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by IL-1beta might be, at least in part, mediated through activation of the Ras/Raf/MEK/MAPK pathway in TSMCs.

Interleukin-1β enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth-muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway

Elevated levels of several cytokines including interleukin-1β (IL-1β) have been detected in airway fluid of asthmatic patients. Inhalation of IL-1β induced a bronchial hyper-reactivity to contractile agonists. However, the implication of IL-1β in the pathogenesis of bronchial hyper-reactivity is not completely understood. Therefore, we investigated the effect of IL-1β on bradykinin (BK)-induced inositol phosphate [Ins(X)P] accumulation and Ca2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth-muscle cells (TSMCs). Treatment of TSMCs with IL-1β potentiated BK-induced Ins(X)P accumulation and Ca2+ mobilization. However, there was no effect on the Ins(X)P response induced by endothelin-1, 5-hydroxytryptamine or carbachol. Treatment with platelet-derived growth factor B-chain homodimer (PDGF-BB) also enhanced the BK-induced Ins(X)P response. These enhancements by IL-1β and PDGF-BB might be due to an up-regulation of BK B2 receptor density (Bmax), since [3H]BK binding to TSMCs was inhibited by the B2-selective agonist and antagonist, BK and Hoe 140, but not by B1-selective reagents. The enhancing effects of IL-1β and PDGF-BB on Ins(X)P accumulation, Ca2+ mobilization and Bmax were attenuated by PD98059 [an inhibitor of activation of mitogen-activated protein kinase (MAPK) kinase, MEK] and cycloheximide (an inhibitor of protein synthesis), suggesting that IL-1β may share a common signalling pathway with PDGF-BB via protein synthesis. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed the up-regulation of BK receptors induced by IL-1β, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by IL-1β might be, at least in part, mediated through activation of the Ras/Raf/MEK/MAPK pathway in TSMCs.

Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcription-promoting activity.

Lipopolysaccharide enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells.

Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen-activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)-induced inositol phosphates (IPs) accumulation and Ca(2+) mobilization in canine cultured tracheal smooth muscle cells (TSMCs). LPS stimulated phosphorylation of p42/p44 MAPK in a time- and concentration-dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min-incubation and the maximal effect was achieved with 100 microg ml(-1) LPS. Pretreatment of TSMCs with LPS potentiated BK-induced IPs accumulation and Ca(2+) mobilization. However, there was no effect on the IPs response induced by endothelin-1, 5-hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF-BB enhanced BK-induced IPs response. These enhancements by LPS and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)) in TSMCs, characterized by competitive inhibition of [(3)H]-BK binding using B(1) and B(2) receptor-selective reagents. The enhancing effects of LPS and PDGF-BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF-BB in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Tumour necrosis factor-alpha- and interleukin-1beta-stimulated cell proliferation through activation of mitogen-activated protein kinase in canine tracheal smooth muscle cells.

The elevated levels of inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF-alpha and IL-1beta on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. TNF-alpha and IL-1beta induced [(3)H]-thymidine incorporation in a time- and concentration-dependent manner. The maximal stimulation of [(3)H]-thymidine incorporation induced by TNF-alpha and IL-1beta was seen 12 h after incubation with these cytokines. In response to TNF-alpha and IL-1beta, p42/p44 MAPK was activated with a concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF-alpha and IL-1beta. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)-phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)-PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. TNF-alpha- and IL-1beta-induced [(3)H]-thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF-alpha and IL-1beta were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF-alpha- and IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in TSMCs.

Mechanisms underlying the inhibitory effect of propofol on the contraction of canine airway smooth muscle.

Propofol has been shown to produce relaxation of preconstricted airway smooth muscle. Although the inhibition of calcium mobilization is supposed to be the major mechanism of action, the whole picture of the mechanisms is not completely clear. Contractile response was performed using canine tracheal rings. The effects of propofol on carbachol-induced mobilization of intracellular Ca2+ and phosphoinositide hydrolysis were measured using cultured canine tracheal smooth muscle cells by monitoring fura-2 signal and assessing the accumulation of [3H]-inositol phosphates. To detect the effect of propofol on muscarinic receptor density and affinity, [3H]N-methyl-scopolamine was used as a radioligand for receptor binding assay. Pretreatment with propofol shifts the concentration-response curves of carbachol-induced smooth muscle contraction to the right in a concentration-dependent manner without changing the maximal response. Propofol not only decreased the release of Ca2+ from internal stores but also inhibited the calcium influx induced by carbachol. In addition, carbachol-induced inositol phosphate accumulation was attenuated by propofol; the inhibitory pattern was similar to the contractile response. Moreover, propofol did not alter the density of muscarinic receptors. The dissociation constant value was not altered by pretreatment with 100 microM propofol but was significantly increased by 300 microM (propofol, 952+/-229 pM; control, 588+/-98 pM; P<0.05). Propofol attenuates the muscarinic receptor-mediated airway muscle contraction. The mechanism underlying these effects was attenuation of inositol phosphate generation and inhibition of Ca2+ mobilization through the inhibition of the receptor-coupled signal-transduction pathway.

Inhibitory effects of diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells.

Benzodiazepines have a direct bronchodilator action in airway smooth muscle, but the mechanisms by which these agents produce muscle relaxation are not fully understood. The current study was performed to identify the effects of the benzodiazepines diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells. Whole-cell patch-clamp recording techniques were used to evaluate the effects of the benzodiazepines diazepam (10(-8) to 10(-3) M) and midazolam (10(-8) to 10(-3) M) on inward Ca2+ and outward K+ channel currents in dispersed canine tracheal smooth muscle cells. The effects of the antagonists flumazenil (10(-5) M) and PK11195 (10(-5) M) on these channels were also studied. Each benzodiazepine tested significantly inhibited Ca2+ currents in a dose-dependent manner, with 10(-6) M diazepam and 10(-5) M midazolam each causing approximately 50% depression of peak voltage-dependent Ca2+ currents. Both benzodiazepines promoted the inactivated state of the channel at more-negative potentials. The Ca2+-activated and voltage-dependent K+ currents were inhibited by diazepam and midazolam (> 10(-5) M and > 10(-4) M, respectively). Flumazenil and PK11195 had no effect on these channel currents or on the inhibitory effects of the benzodiazepines. Diazepam and midazolam had inhibitory effects on voltage-dependent Ca2+ channels, which lead to muscle relaxation. However, high concentrations of these agents were necessary to inhibit the K+ channels. The lack of antagonized effects of their antagonists is related to the non-gamma-aminobutyric acid-mediated electrophysiologic effects of benzodiazepines on airway smooth muscle contractility.

Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes.

We tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to reacquire the abundant contractile protein content, marked shortening capacity, and elongated morphology characteristic of contractile cells within intact tissue. Passage 1 or 2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days. During serum deprivation, two differentiation pathways emerged. One-sixth of the cells developed an elongated morphology and aligned into bundles. Elongated myocytes contained cables of contractile myofilaments, dense bodies, gap junctions, and membrane caveoli, ultrastructural features of contractile SM in tissue. These cells immunostained intensely for SM alpha-actin, SM myosin heavy chain (MHC), and SM22 (an SM-specific actin-binding protein), and Western analysis of culture lysates disclosed 1.8 (SM alpha-actin)-, 7.7 (SM MHC)-, and 5.8 (SM22)-fold protein increases during serum deprivation. Immunoreactive M3 muscarinic receptors were present in dense foci distributed throughout elongated, SM MHC-positive myocytes. ACh (10(-3) M) induced a marked shortening (59.7 +/- 14.4% of original length) in 62% of elongated myocytes made semiadherent by gentle proteolytic digestion, and membrane bleb formation (a consequence of contraction) occurred in all stimulated cells that remained adherent and so did not shorten. Cultured airway myocytes that did not elongate during serum deprivation instead became short and flattened, lost immunoreactivity for contractile proteins, lacked the M3 muscarinic-receptor expression pattern seen in elongated cells, and exhibited no contractile response to ACh. Thus we demonstrate that prolonged serum deprivation induces distinct differentiation pathways in confluent cultured tracheal myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.

Dissociation of Intracellular Ca 2+ Release and Ca 2+ Entry Response to 5-Hydroxytryptamine in Cultured Canine Tracheal Smooth Muscle Cells

The relationship between the agonist-sensitive Ca 2+ pool and those discharged by the Ca 2+-ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca 2+ ([Ca 2+] i), followed by a sustained plateau phase that was dependent on extracellular Ca 2+. In such cells, TG produced a concentration-dependent increase in [Ca 2+] i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca 2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca 2+] i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca 2+-ATPase inhibitors, cyclopiazonic acid and 2,5-di- t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca 2+-influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca 2+ stores is sufficient for activation of Ca 2+ influx. Some characteristics of the Ca 2+-influx activated by depletion of internal Ca 2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca 2+ influx was inhibited by La 3+, membrane depolarisation, and the novel Ca 2+-influx blocker 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca 2+ influx by TG also was blocked by La 3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca 2+ stores in TSMCs is sufficient for activation of Ca 2+ influx, and (2) the agonist-activated Ca 2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca 2+ pool are indistinguishable.

Effect of forskolin on endothelin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

1. The effects of increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) on endothelin-1 (ET-1)-induced generation of inositol phosphates (IPs) and increase in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). 2. Pretreatment of TSMCs with either cholera toxin (CTX; 10 microg ml(-1), 4 h), forskolin (10 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited ET-1-stimulated Ca2+ mobilization (by 23 +/- 5%, n = 8) and IPs accumulation (by 32 +/- 6%, n = 4). While after treatment with forskolin for 24 h, the cells retained the ability to respond to ET-1-induced Ca2+ mobilization to the same extent as the control group. 3. Forskolin (1-100 microM) inhibited the ET-1-induced increase in [Ca2+]i, but the lower concentrations had little effect on this response. 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 ET-1 without changing the -logEC50 values. 4. The water-soluble forskolin analogue L-858051, 7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin, significantly inhibited ET-1-stimulated IPs accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive analogue of forskolin, had little effect on stimulated responses. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit ET-1-induced IPs accumulation. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. 5. The locus of this inhibition of forskolin treatment on AlF4(-)-stimulated IPs accumulation was investigated in canine TSMCs. The AlF4(-)-induced IPs accumulation was inhibited by forskolin, supporting that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. 6. We conclude that cAMP elevating agents inhibit ET-1-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of ET-1 receptors, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.

A1115 The Inhibitory Effects of Benzodiazepines on Voltage-dependent Ca sup 2+ and K sup + Channels in Canine Tracheal Smooth Muscle Cells

A1115 The Inhibitory Effects of Benzodiazepines on Voltage-dependent Ca sup 2+ and K sup + Channels in Canine Tracheal Smooth Muscle Cells

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.

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.