Pretreatment Of Tracheal Smooth Muscle Cells Research Articles

Thrombin-stimulated cell proliferation mediated through activation of Ras/Raf/MEK/MAPK pathway in canine cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients and shown to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). However, the implication of thrombin in the cell proliferation was not completely understood. In this study, thrombin stimulated [ 3H]thymidine incorporation and p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [ 3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C inhibitor GF109203X, removal of Ca 2+ by addition of BAPTA/AM plus EGTA, PI 3-kinase inhibitors wortmannin and LY294002, and inhibitor of MEK1/2 PD98059. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca 2+, PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in canine cultured TSMCs.

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.

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [ 3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [ 3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca 2+ by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [ 3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca 2+, PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.

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.

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.

Inhibition of 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester.

1. Regulation of the increase in inositol-1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5-hydroxytryptamine (5-HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. 2. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the 5-HT-induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5-HT-induced Ca2+ mobilization reached the same extent as control cells. 3. The concentration of PMA that gave half-maximal inhibition of 5-HT-induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 microM) of GF109203X (0.1 microM), PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. In parallel with the effect of PMA on 5-HT-induced IP3 formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC-alpha, beta I, beta II, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down-regulation of these PKC isozymes PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. In conclusion, these results suggest that activation of PKC may inhibit the receptor-mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta induced by PMA caused an attenuation of 5-HT-stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.

Effect of Forskolin onBradykinin-Induced Calcium Mobilizationin Cultured Canine Tracheal Smooth Muscle Cells

The effects of increases in intracellular adenosine 3′:5′-cyclic monophosphate (cyclic AMP) on bradykinin (BK)-induced generation of inositol phosphates (IPs) and Ca 2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with either forskolin or dibutyryl cyclic AMP attenuated BK-stimulated responses. The inhibitory effects of these agents produced both a depression of the maximal response and a shift to the right of the concentration-response curves of BK. The water-soluble forskolin analogue L-858051, 7-deacetyl-7β-(r-N-methylpiperazino)-butyryl forskolin, significantly attenuated BK-stimulated IPs accumulation, while 1,9-dideoxy forskolin, an inactive forskolin, had little effect on IPs 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-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cyclic AMP-dependent protein kinase (PKA), reversed the ability of forskolin to attenuate BK-stimulated IPs accumulation. The K D and B max values of the BK receptor for [ 3H]BK binding were not significantly changed by forskolin treatment for 30 min and 4 h. The AlF − 4-induced IPs accumulation was attenuated by forskolin, indicating that G protein(s) are directly activated by AlF − 4 and uncoupled to phospholipase C by forskolin treatment. These results suggest that activation of cyclic AMP/PKA might inhibit the BK-stimulated PI breakdown and consequently reduce the [Ca 2+] i increases or inhibit independently both responses, which is distal to the BK receptor in canine cultured TSMCs.

Muscarinic inhibition of L-type Ca2+ channels in guinea-pig tracheal smooth muscle cells.

Modulation of L-type Ca2+ channels by acetylcholine (ACh) was studied in enzymatically isolated guinea-pig tracheal smooth muscle cells (TSMCs). ACh reversibly inhibited whole cell L type Ca2+ current measured with Ba2+ ions as charge carriers (I(Ba)). With pipette solution containing 0.1 mM EGTA, 1 microM ACh induced transient inhibition of I(Ba) followed by sustained inhibition (67.0+/-3.7% of the control, n=19). When intracellular Ca2+ concentration ([Ca2+]i) was fixed at 50 nM by BAPTA-Ca2+ buffer in the pipette, the transient inhibition was abolished whereas the sustained inhibition (66.0+/-7.8%, n=6) still occurred, suggesting that the transient inhibition was attributed to inactivation of the channels induced by increase in [Ca2+]i. The sustained inhibition was abolished when [Ca2+]i was fixed at zero. The sustained inhibition of I(Ba) by 1 microM ACh was observed in the presence of 10 microM AF-DX 116, whereas it was not observed in the presence of 1 microM 4 DAMP. ACh did not inhibit I(Ba) in the presence of 1 mM GDP-beta-S in the pipette, whereas the drug irreversibly inhibited the current in the presence of 0.1 mM GTP-gamma-S in the pipette. Pretreatment of TSMCs with pertussis toxin did not altered the effects of ACh. Application of neither 1-oleoyl-2-acetyl sn-glycerol (1 microM) nor phorbol 12-myristate 13-acetate (1 microM) reduced I(Ba). These results suggest that the sustained inhibition of I(Ba) by ACh is mediated by Ca2+ requiring and protein kinase C-independent mechanisms existing in the downstream of G-protein coupled with M3 receptors.

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.

Inhibitory Effect of Phorbol Ester on Carbachol-Induced Signal Transduction in Cultured Canine Tracheal Smooth Muscle Cells.

Regulation of the increases in inositol 1,4,5-trisphosphate (IP(3)) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP(3) formation and caused an initial transient peak of [Ca(2+)](i) followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 &mgr;M) for 30 min blocked the carbachol-induced IP(3) formation and Ca(2+) mobilization. Following preincubation, carbachol-induced Ca(2+) mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP(3) formation and increase in [Ca(2+)](i) were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 &mgr;M), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 &mgr;M, did not inhibit these responses to carbachol. The K(d) and B(max) of the muscarinic receptor for [(3)H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity. Copyright 1995 S. Karger AG, Basel

Inhibitory effect of phorbol ester on bradykinin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

Regulation of the increase in inositol 1,4,5-trisphosphate (IP 3) production and intracellular Ca 2+ concentration ([Ca 2+] i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by bradykinin (BK)led to IP 3 formation and caused an initial transient peak followed by a sustained elevation of [Ca 2+] i in a concentration-dependent manner. Pretreatment of TSMCs with phorbo112-myristate 13-acetate (PMA,1 μM) for 30 min blocked the BK-induced IP 3 formation and Ca 2+ mobilization. However, this inhibition was reduced after incubating the cells for 4 h with PMA. Inactive phorbol ester, 4α-phorbol 12,13-didecanoate at 1 μM, did not inhibit these responses to BK. Prior treatment with staurosporine (1 μM), a PKC inhibitor, inhibited the effect of PMA on the BK-induced response, suggesting that the effect of PMA is mediated by the activation of PKC. In parallel experiments, a change of PKC activity was observed. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the cell membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24h of PMA treatment. Moreover, treatment with 1 uM PMA for 2 and 24 h did not significantly change the KD and B max of the BK receptor for [ 3H]BK binding (control: K D = 2.3 ± 0.3 nM, B max = 25.2 ± 1.4 fmol/mg protein). These results suggest that activation of PKC inhibit IP 3 accumulation and consequently attenuate [Ca 2+] i increase or inhibit independently both responses. The PMA-induced inhibition of responses to BK was associated with an increase in membranous PKC activity.

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization induced by endothelin-1 in cultured canine tracheal smooth muscle cells

Regulation of the increase in inositol 1,4,5-trisphosphate (IP 3) production and intracellular Ca 2+ concentration ([Ca 2+] i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by endothelin-1 (ET-1) led to IP 3 formation and caused an initial transient peak followed by a sustained elevation of [Ca 2+] i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 μM ) for 30 min blocked the ET-1-induced IP 3 formation and Ca 2+ mobilization. However, this inhibition was reduced after incubating the cells for 8 h with PMA. Following preincubation, ET-1-induced Ca 2+ mobilization recovered with time and reached the same extent of control cells within 48 h. The concentrations of PMA that gave half-maximal inhibition (-IogEC 50) of ET-1-induced IP 3 formation and increase in [Ca 2+] i were 8.6 and 8.4 M, respectively. Prior treatment of TSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate ET-1-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the ET-1-induced IN formation and Ca 2+ mobilization, a change of PKC activity was observed in TSMCs. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca 2+] i increase or inhibit independently both responses. The PMA-induced inhibition of responses to ET-1 was associated with an increase in membranous PKC activity.

Endothelin‐ and Sarafotoxin‐Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells

The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphatase (IPs) and tracheal smooth muscle contraction. BQ-123, an ETA receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pKB values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca2+ and was blocked by treatment with EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca(2+)-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 microM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to downregulation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

5‐Hydroxytryptamine receptor‐mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor. 7. Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine.8. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin- and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HT stimulated contraction of tracheal smooth muscle.

Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells.

1. Stimulation of bradykinin (BK) receptors coupled to phosphoinositide (PI) hydrolysis was investigated in canine cultured tracheal smooth muscle cells (TSMCs). BK, kallidin, and des-Arg9-BK, stimulated [3H]-inositol phosphates (IPs) accumulation in a dose-dependent manner with half-maximal responses (EC50) at 20 +/- 5, 13 +/- 4, and 2.3 +/- 0.7 nM, (n = 5), respectively. 2. D-Arg[Hyp3, D-Phe7]-BK and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK, B2 receptor antagonists, were equipotent in blocking the BK-induced IPs accumulation with pKB = 7.1 and 7.3, respectively. 3. Short-term exposure of TSMCs to phorbol 12-myristate 13-acetate (PMA, 1 microM) attenuated BK-stimulated IPs accumulation. The concentrations of PMA that gave half-maximal and maximal inhibition of BK-induced IPs accumulation were 15 +/- 4 nM and 1 microM, n = 3, respectively. The inhibitory effect of PMA on BK-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. Prolonged incubation of TSMCs with PMA for 24 h, resulted in a recovery of receptor responsiveness which may be due to down-regulation of PKC. The inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit this response. 5. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the G protein(s) can be directly activated by AlF4-, which is uncoupled from phospholipase C by PMA treatment. 6. Incubation of TSMCs in the absence of external Ca2+ or upon removal of Ca2+ by addition of EGTA, caused a decrease in IPs accumulation without changing the basal levels. Addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs stimulated IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or BK. The combination of GTP gamma S and BK caused an additive effect on IPs accumulation.7. Pretreatment of TSMCs with cholera toxin enhanced BK-stimulated IPs accumulation, whereas there was no effect with pertussis toxin.8. These data suggest that BK-stimulated PI metabolism is mediated by the activation of BK B2 receptors coupling to a G protein which is not blocked by cholera toxin or pertussis toxin treatment and dependent on external Ca2+. The transduction mechanism of BK coupled to PI hydrolysis is sensitive to feedback regulation by PKC.

Primary culture of canine tracheal smooth muscle cells in serum-free medium: effects of insulin-like growth factor I and insulin.

The effects of growth factors on cell growth and muscarinic receptor (mAChR) expression of canine tracheal smooth muscle cells (TSMCs) were observed under serum-free medium supplemented with 0.1% BSA. In the presence of 0.1% BSA, TSMCs withdraw from cell cycle as compared with 10% FBS and allow to determine the effects of growth factors on mAChR expression. The individual components of growth factors (IGF-I, insulin, and aFGF) at the concentration used are not sufficient to stimulate growth of TSMCs in the primary culture with 0.1% BSA. IGF-I (10 ng/ml) and insulin (1 microgram/ml), alone or in combination, could stimulate the expression of mAChRs of cultured TSMCs. Heparin could inhibit these stimulatory effects of mAChR expression. The stimulatory effects of IGF-I and insulin on mAChR expression were mediated through their own receptors since these effects were reversed by pretreatment of TSMCs with antibodies of the respective growth factor receptors. The pharmacological response of functional mAChRs, determined as accumulation of inositol phosphates induced by carbachol, is greater in the medium containing IGF-I and insulin than that cultured in 0.1% BSA. These results firmly establish that IGF-I and insulin could stimulate the expression of mAChRs in TSMCs under serum-free culture condition.