Introduction

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We recently demonstrated that the treatment of tracheal smooth muscle strips with the cholinergic agonist acetylcholine (ACh) activates phospholipase D (PLD; Mamoon et al., 1999). The increase in PLD activity elicited by ACh treatment appeared to be both protein kinase (PK) C dependent and independent of PKC activity. Other investigators have reported that the treatment of Rat-1 fibroblasts with forskolin also activates PLD (Ruan et al., 1998). Forskolin directly activates adenylyl cyclase and increases the intracellular concentration of cAMP, and hence increases PKA activity (Laurenza et al., 1989). Studies by Ginsberg et al. (1997) suggested that the PKC- and PKA-dependent signal transduction pathways converge in the activation of PLD and that the stimulation of both pathways was required for optimal physiological activation of PLD in thyroid cell line FRTL-5 by thyroid-stimulating hormone.

Activation of muscarinic acetylcholine receptor (mAChR) generates second messengers via the phosphoinositide and adenylyl cyclase systems (Caulfield, 1993). Expression models have demonstrated that M1, M3, and M5 subtypes of mAChR can activate multiple signaling effectors including PLC, phospholipase A₂, and phospholipase D (PLD; Felder, 1995). Muscarinic M2 and M4 receptors are also coupled to the activation of PLD and phospholipase A₂ (Felder, 1995). Definitive activation of PLD as a consequence of treatment with mAChR agonists was shown using human embryonic kidney cells transfected with the M1, M2, M3, or M4 receptors (Sandmann et al., 1991). It was established that the activation of all four mAChR subtypes (M1, M2, M3, and M4) increases PLD activity but that M1 and M3 receptors coupled to PLD activation with higher efficiency than did M2 and M4 receptors (Sandmann et al., 1991). Tracheal smooth muscle cells have approximately 80% M2 and 20% M3 receptors (Caulfield, 1993), both of which may activate PLD in this tissue.

An interaction between ₂-adrenoceptors and muscarinic receptors on the adenylyl cyclase system has been demonstrated by a number of investigators (Houslay, 1991; Kotlikoff and Kamm, 1996). It has been shown that activation of ₂-adrenoreceptors inhibits the increase in intracellular Ca²⁺ caused by muscarinic receptor activation and inhibits phosphorylation of myosin light chains by the muscarinic agonists (Kotlikoff and Kamm, 1996). Although stimulation of PLD by adrenoceptors and muscarinic receptor agonists has been reported, little is known about the possible interaction between the cAMP pathway and the mAChR-induced PLD pathway. This interaction might help to explain the mechanisms involved in the relaxation of mAChR-induced contraction by cAMP agonists. In the present study, we investigated the stimulation of PLD by forskolin, a compound that directly increases adenylyl cyclase activity, and isoproterenol, a ₂-adrenoceptor agonist. The role of PKA or PKC in forskolin- or isoproterenol-induced PLD activation and the interaction of ₂-adrenoceptor activation or elevated cAMP- and ACh-induced PLD activity in intact porcine tracheal smooth muscle cells were evaluated.

	Experimental Procedures

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Materials. [9,10-³H(N)]Palmitic acid, [³²P]orthophosphate, and myo-[³H]inositol were obtained from DuPont-New England Nuclear (Boston, MA). ACh chloride, atropine, and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma Chemical Co. (St. Louis, MO). Bisindolylmaleimide I (GFX), calphostin C, H-89, KT5720, isoproterenol hydrochloride, and forskolin were obtained from Calbiochem Corp. (La Jolla, CA). Phosphatidylethanol (PEth) standard was purchased from Avanti Polar Lipids (Alabaster, AL). Anti-PLC-₂ antibody, protein G PLUS-Agarose, and molecular weight markers were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SuperSignal west dura extended duration substrate (Luminol) was obtained from Pierce Chemical Co. (Rockford, IL). All other materials were from Sigma Chemical Co. Animals were purchased from local suppliers.

Preparation of Muscle Strips. Male Yorkshire pigs (weight, 20-30 kg) were anesthetized with 5% isoflurane in 2 liters/min O₂ and sacrificed by exsanguination. The trachea was quickly removed, irrigated with 0.9% NaCl, and transferred to a sterile container containing normal external solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 5.5 mM glucose, 0.5 ml penicillin-streptomycin, and 10 mM HEPES buffer, pH adjusted to 7.4). The trachea was cut open longitudinally, and smooth muscle was dissected free of epithelium, gland cells, connective tissue, and cartilage. The smooth muscle was then carefully cut into segments of single-ring width. Muscle strips were maintained at 37°C in an atmosphere containing 5% CO₂ in Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, and 12 mM glucose, pH 7.4).

[³H]Palmitic Acid Labeling of Muscle Strips and Incubation Conditions. Tracheal smooth muscle segments (1 ring) were incubated for 6 h at 37°C in 3 ml of Krebs-Henseleit buffer with 3 µCi/ml [³H]palmitic acid to label phospholipids. At the end of the incubation period, tissue was washed three times with Krebs-Henseleit buffer and then transferred to test tubes containing 1 ml of the same wash buffer. ACh and atropine were diluted with Krebs-Henseleit buffer. Forskolin, PMA, GFX, calphostin C, H-89, and KT5720 were dissolved in DMSO and then diluted with buffer to final working concentrations. Control strips were treated with equal amounts of dimethyl sulfoxide (0.01-0.1%). Ethanol was added 10 min before the addition of the pharmacological agents or vehicle. The incubation conditions for each experiment are given in the figure legends.

Lipid Extraction. Lipids were extracted according to a modified Bligh and Dyer (1959) procedure. Briefly, incubations were stopped by the addition of 4 ml of methanol containing 2% acetic acid to each sample. Methylene chloride (2 ml) was added, and the sample was homogenized and shaken vigorously. Samples were allowed to stand at room temperature for 30 to 60 min. An additional 2 ml of methylene chloride and 2 ml of 1 M KCl were then added. The organic and aqueous phases were separated by centrifugation, after which the bottom phase was collected, and solvent was removed under a stream of nitrogen. The extracted material was redissolved in 100 µl of methylene chloride/methanol (1:1) and stored at 20°C.

Thin-Layer Chromatography (TLC) and Quantification. PEth was separated using one-dimensional TLC. Aliquots of 20 µl were spotted onto the TLC plates, and the plates were developed using the upper phase of a solvent system consisting of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (13:2:3:10). Radioactive phospholipids were located on the plate using a beta emission scanner (Bioscan Inc., Washington, DC) and by comparing the position of radioactive material to the position of phosphatidic acid or PEth standards that were run on the same plate. Areas corresponding to PEth standards were scraped from the plates, and the radioactivity was measured by scintillation spectroscopy.

Identification and Phosphorylation Assay for PLC-₂ Isoform. The PLC-₂ isoform in tracheal smooth muscle was identified by using a protocol supplied with the antibody by Santa Cruz Biotechnology. In short, after stopping the incubations, muscle strips were cut into small pieces, homogenized in 3 ml of RIPA buffer with inhibitors (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate. 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin, and 100 mM sodium orthovanadate) using a Polytron device, and incubated on ice for 10 min. Cellular debris were pelleted by centrifugation at 5000 rpm at 4°C for 15 min. Supernatants were then transferred to conical centrifuge tubes, and 20 µl of protein G PLUS-Agarose was added to each tube and incubated at 4°C for 30 min. Samples were then centrifuged at 2500 rpm at 4°C for 5 min, and 1 ml of the supernatant was collected in microcentrifuge tubes. Primary antibody (2 µg) was then added to each tube and incubated for 1 h at 4°C. Protein G PLUS-Agarose (20 µl) was added to each sample and incubated at 4°C for 1 h on a rotating device. Immunoprecipitates were then collected via centrifugation at 3000 rpm for 5 min. Pellets were washed four times with PBS and, after the final wash, were resuspended in 40 µl of electrophoresis sample buffer and boiled for 3 min. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were washed three times with Tris-buffered saline with 0.05% Tween-20 and then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody (anti-rabbit Ig), diluted to 1:20,000. Nonspecific binding was blocked by 5% milk (w/v) in Tris-buffered saline, pH 8.0 with 0.05% Tween-20. Proteins were then visualized using an enhanced chemiluminescence detection kit (Luminol). For phosphorylation assays, muscle strips were prelabeled with [³²P]orthophosphate (1 mCi/ml) for 6 h. Labeled cell lysates containing immunoprecipitated proteins were collected as described above and separated on a 10% SDS-polyacrylamide gel. Gels were dried, and phosphorylated proteins were quantified by placing the gel onto a storage phosphor screen for 6 h and then scanning the exposed plate with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Extraction and Analysis of Radiolabeled Inositol-1,4,5-Trisphosphate (IP₃). IP₃ was extracted according to a modified procedure described by Godfrey (1992). Muscle strips (three rings wide) were prelabeled with myo-[³H]inositol (5 µCi/ml) for approximately 36 h in Dulbecco's modified Eagle's medium nutrient mixture with Ham's F-12. At the end of labeling, strips were washed three times with normal external solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 10 mM HEPES, and 5.5 mM glucose, pH 7.4) and then incubated in 1 ml of the same buffer in a shaking water bath at 37°C for approximately 10 min before the addition of drugs. Incubations were terminated with 30% ice-cold trichloroacetic acid (200 µl), and tissues were disrupted with a Polytron device and centrifuged at 5000 rpm for 10 min. Then, 1 ml of supernatant was collected in glass test-tubes, and 1 ml of water-saturated ether was added. Tubes were placed in a methanol/dry-ice bath to freeze the aqueous sample, and ether was poured off. Ether extraction was repeated three times. The aqueous extract containing the inositol phosphates was then diluted to 10 ml with water and loaded onto columns filled with 2 ml of Dowex AG1X8 anion exchange resin (200-400 mesh, formate form). Free inositol was eluted with 20 ml of water, inositol monophosphates were then eluted with 16 ml of 0.2 M ammonium formate/0.1 M formic acid, and inositol bisphosphates were eluted with 16 ml of 0.4 M ammonium formate/0.1 M formic acid. Finally, inositol trisphosphates were eluted with 8 ml of 0.8 M ammonium formate/0.1 M formic acid and collected in 15-ml tubes. Scintillant (4 ml) was added to 1 ml fractions of the eluents and counted for radioactivity.

Protein Assay. Protein levels were estimated according to the method of Lowry et al. (1951) using prepared reagents (Pierce Chemical Co.) and BSA as the standard

Data Presentation. Data from three or more treatment groups or multiple treatment of the same group were compared using one-way or repeated measures ANOVA, respectively. All data were expressed as mean ± S.E. and were collected from at least three experiments using three different animals. Data were deemed significant when P  .05. All statistical analyses were performed using Primer of Biostatistics Software (Version 4.0).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Strips of smooth muscle were labeled with [³H]palmitic acid, and the activity of PLD was measured by using the formation of the transphosphatidylation product, [³H]PEth. Figure 1 shows time-dependent increases in forskolin- and isoproterenol-induced PLD activation. Stimulation of the muscle strips with forskolin (10⁵ M) in the presence of 100 mM ethanol caused a significant increase (179% versus control, P < .01) in [³H]PEth formation in 5 min and reached a plateau at 20 min. However, isoproterenol (10⁶ M)-induced [³H]PEth formation was slower with a significant increase (118% versus control, P < .01) in [³H]PEth formation occurring after 10 min. Like forskolin, stimulation of PLD by isoproterenol was complete in 20 min. Figure 2 shows the effects of increasing concentrations of forskolin and isoproterenol on PLD activation. All observations were made at 20 min. Forskolin (10⁷ M) increased PLD activity by 178% (P < .01). A similar concentration of isoproterenol (10⁷ M) caused a 98% increase (P < .05) in [³H]PEth formation. Maximum stimulation of PLD activity was achieved by 10⁵ M forskolin (482% versus control, P < .001) and 10⁶ M isoproterenol (304% versus control, P < .001). Higher concentrations of either agonist failed to further increase the formation of [³H]PEth.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Time course of forskolin- and isoproterenol-stimulated [3H]PEth formation. [3H]Palmitic acid-labeled muscle strips were preincubated with 100 mM ethanol for 10 min before treatment with vehicle, forskolin (105 M), or isoproterenol (106 M) for times indicated. Results are given as mean ± S.E. of three experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of increasing concentration of forskolin or isoproterenol on [3H]PEth formation. [3H]Palmitic acid-labeled muscle segments were preincubated with 100 mM ethanol for 10 min before they were exposed to increasing concentrations of forskolin (108-104 M) or isoproterenol (108-104 M). Incubations were stopped after 20 min. Values are presented as mean ± S.E. of triplicate determinations for each group.

To determine whether forskolin- or isoproterenol-induced increases in PLD activity were mediated via PKA, muscle strips were pretreated with two different PKA inhibitors: H-89 (10⁷ M) and KT5720 (10⁷ M). Concentrations for the inhibitors were chosen according to previous reports (Findik et al., 1995). These inhibitors become "nonspecific" only at high micromolar concentrations. H-89 and KT5720 reduced forskolin-induced PLD activity by 55% (P < .05) and 58% (P < .05), respectively (Fig. 3A). In a similar experiment, isoproterenol-induced PLD activity was reduced 40% (P < .05) and 37% (P < .05) by H-89 and KT5720, respectively (Fig. 3B). To determine whether PKC plays a role in forskolin- or isoproterenol-mediated increases in PLD activity, forskolin- and isoproterenol-induced PLD activation were studied in muscle strips pretreated with PKC inhibitors calphostin C (10⁶ M) or GFX (10⁶ M). Figure 4 shows that neither calphostin C nor GFX affected forskolin- or isoproterenol-induced [³H]PEth formation. Direct stimulation of PKC with the phorbol ester PMA (10⁸ M), activated PLD. This activation was inhibited in the presence of calphostin C (10⁶ M) but not in the presence of H-89 (10⁷ M; Fig. 5).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Effect of PKA inhibitors H-89 and KT5720 on forskolin (For; 105 M)- and isoproterenol (Iso; 106 M)-induced [3H]PEth formation. [3H]Palmitic acid-labeled muscle segments were preincubated in the presence of 100 mM ethanol for 10 min before incubation with H-89 (107 M), KT5720 (107 M), forskolin (105 M; A), or isoproterenol (106 M; B) for 20 min. Inhibitors were added to appropriate groups 10 min before the addition of forskolin or isoproterenol. Values are presented as mean ± S.E. of triplicate determinations for each group from three different experiments. *P < .05 compared with forskolin- or isoproterenol-treated groups.

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Effect of PKC inhibitors GFX and calphostin C on forskolin (For; 105 M)- and isoproterenol (Iso; 106 M)-induced [3H]PEth formation. [3H]Palmitic acid-labeled muscle segments were preincubated in the presence of 100 mM ethanol for 10 min before incubation with GFX (106 M), calphostin C (Cal C; 106 M), forskolin (105 M; A), or isoproterenol (106 M; B) for 20 min. Inhibitors were added to appropriate groups 10 min before the addition of forskolin or isoproterenol. Values are presented as mean ± S.E. of triplicate determinations for each group from three different experiments.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Effect of PKA inhibitor H-89 and PKC inhibitor calphostin C (Cal C) on PMA-induced [3H]PEth formation. [3H]Palmitic acid-labeled muscle segments were preincubated in the presence of 100 mM ethanol for 10 min before incubation with PMA (108 M), H-89 (107 M), or calphostin C (106 M) for 20 min. Inhibitors were added to appropriate groups 10 min before the addition of PMA. Values are presented as mean ± S.E. of triplicate determinations for each group from three different experiments. *P < .05 compared with PMA-treated group.

We have previously shown that ACh transiently increases PLD activity in tracheal smooth muscle strips (Mamoon et al., 1999) in both a PKC-dependent and a PKC-independent manner. To determine whether forskolin or isoproterenol has any effect on ACh-induced PLD activity in tracheal smooth muscle cells, muscle strips were treated with ACh (10⁵ M) alone or in combination with either forskolin (10⁵ M) or isoproterenol (10⁶ M). Both forskolin (P < .01; Fig. 6A) and isoproterenol (P < .01; Fig. 6B) inhibited the initial activation of PLD by ACh within the first 5 min. However, there was still an increase in [³H]PEth formation that occurred with a time course similar to that of forskolin or isoproterenol alone, with no significant difference at 20 min. In other experiments, cAMP agonists were used in combination with the PKA inhibitor H-89 to evaluate whether inhibition of ACh-induced PLD activation is PKA dependent in tracheal smooth muscle cells. The PKA inhibitor completely eliminated the effects of forskolin and isoproterenol on ACh-induced PLD activation. Forskolin had no effect on PMA-induced PLD stimulation. The results are given in Table 1.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of coadministration of forskolin and ACh or isoproterenol and ACh on [3H]PEth formation. [3H]Palmitic acid-labeled muscle segments were preincubated in the presence of 100 mM ethanol for 10 min before incubation with ACh (105 M), forskolin (105 M), and ACh (105 M) plus forskolin (105 M; A) for times indicated. B, effect of ACh (105 M), isoproterenol (106 M), and ACh (105 M) plus isoproterenol (106 M). Values are presented as mean ± S.E. of triplicate determinations for each group from three different experiments.

View this table: [in this window] [in a new window]   TABLE 1 Effect of forskolin or isoproterenol on Ach-induced PLD activity. [3H]Palmitic acid-labeled tracheal smooth muscle strips were preincubated in the presence of 100 mM ethanol for 10 min before incubation with 105 M ACh, 105 M ACh + 105 M forskolin, 105 M ACh + 106 M isoproterenol, 108 M PMA, or 108 M PMA + 105 M forskolin for 5 min. Where appropriate, the PKA antagonist H-89 was added at least 10 min before the addition of agonists. Values are presented as mean ± S.E. of triplicate determinations for each group.

One possible site for PKA-mediated inhibition of ACh-activation of PLD would be through inhibition of the activity of PLC, probably the ₂ isoform. To test this, the effects of forskolin (10⁵ M) or isoproterenol (10⁶ M) on ACh-induced production of IP₃ were measured. In our experiments, ACh induced an approximately 4-fold rise in IP₃ production within the first 15 s (Fig. 7). This was followed by a significant decline in IP₃ production at 1 and 5 min. Neither forskolin (3498 ± 487 cpm/mg protein) nor isoproterenol (3565 ± 398 cpm/mg protein) had any effect on IP₃ production compared with the untreated group (3400 ± 356 cpm/mg protein). However, significant inhibition of approximately 50% in IP₃ production was achieved by both forskolin (P < .05) and isoproterenol (P < .05) at 15 s, 1 min, and 5 min when coincubated with ACh (1 µM). In addition, the phosphorylation of PLC-₂ via activation of PKA was measured. The PLC-₂ isoform was identified by immunoprecipitation/Western blotting in tracheal smooth muscle cells. The [³²P]orthophosphate-prelabeled muscle strips were used to determine whether forskolin (10⁵ M) or isoproterenol (10⁶ M) could induce phosphorylation of the PLC-₂ band identified by immunoprecipitation/Western blotting. Figure 8 shows that both forskolin and isoproterenol caused an increased phosphorylation of the PLC-₂ isoform at 5 min in tracheal smooth muscle cells. Densitometric measurements revealed that intensities in PLC-₂ bands increased by 420 to 570% and 365 to 525% (compared with column A, untreated control) for forskolin- and isoproterenol-treated samples, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effect of forskolin or isoproterenol on ACh-induced IP3 production. Muscle strips were prelabeled with myo-[3H]inositol for 36 h and then incubated in the presence or absence of various agents as indicated. Incubations were terminated after 15 s, 1 min, and 5 min as described in the text. Values are presented as mean ± S.E. of triplicate determinations for each group from three different experiments. *P < .05 compared with ACh-treated group.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Phosphorylation of PLC-2 by forskolin or isoproterenol and identification of PLC-2 by Western blotting. To determine phosphorylation of PLC-2, [32P]orthophosphate prelabeled muscle strips were incubated (as described in the text) in the presence or absence of forskolin (105 M) or isoproterenol (106 M) for 5 min, and then immunoprecipitated proteins (15 µl/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis, Phosphorylated proteins were revealed by scanning the gel with the PhosphorImager (A-C). D, to identify PLC-2 in tracheal smooth muscle, immunoprecipitated proteins from cell lysates were separated on a 10% SDS-polyacrylamide gel (15 µl/lane) and then detected with enhanced chemiluminescence (described in the text). The position of molecular markers is given in kDa. Each gel is representative of three different experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

PLD activity is measured with a transphosphatidylation reaction (Morris et al., 1997), in which short-chain alcohols can replace water as a PLD substrate and a phosphoester of the alcohol (PEth) is produced rather than phosphatidic acid (Yu et al., 1996). Once formed, PEth is relatively stable and hence is a reliable indicator for PLD activity (Morris et al., 1997). We demonstrate that stimulation of PKA with forskolin or isoproterenol significantly increases PEth levels in tracheal smooth muscle cells in both a time- and concentration-dependent manner. Forskolin-induced PLD activation has been reported in FRTL-5 thyroid cells (Ginsberg et al., 1997); however, in this study a cumulative concentration of PEth at a single time point, 30 min, was used. In tracheal smooth muscle cells, elevation of cAMP takes as little as 5 min to significantly increase the level of PLD activity. The accumulation of PEth continues in a linear fashion for up to 20 min. By way of comparison, muscarinic agonists also stimulate PLD activity in various cells, including tracheal smooth muscle cells (Hosey, 1992; Caulfield, 1993; Mamoon et al., 1999). However, we previously demonstrated that muscarinic agonist-induced PLD activity in this tissue is transient even during continuous stimulation of the muscle by ACh or PMA, reaching maximal activation within 2 min and activity back to basal levels by 5 min (Mamoon et al., 1999).

Both forskolin and isoproterenol activate PKA by raising the intracellular level of cAMP (Seamon and Daly, 1986). The observation that PKA inhibitors block both forskolin- or isoproterenol-induced PLD activity indicates that both of these agents produce their effects, at least in part, via PKA activation. Interestingly, these inhibitors were more effective in reducing forskolin-induced PLD activity (~55% inhibition) than isoproterenol-induced PLD activation (~40% inhibition). The observed difference in the degree of inhibition may be due to the fact that forskolin is a direct activator of the enzyme adenylyl cyclase (Laurenza et al., 1989), whereas isoproterenol is a nonselective -adrenoreceptor agonist (Takemura et al., 1995), which in addition to increasing the intracellular cAMP, activates several other signaling molecules. The activation of PKC in this manner via -adrenoceptor stimulation has been shown to occur and to modulate the ability of -adrenergic agonists to stimulate adenylyl cyclase (Sibley et al., 1984; Houslay, 1991). However, inhibition of PKC activity did not have any effect on either forskolin- or isoproterenol-induced increases in PLD activity in this tissue. It has also been shown that activation of PKC by phorbol esters such as PMA can stimulate PLD activity (Conricode et al., 1992; Kanoh et al., 1992; Mamoon et al., 1999). PMA-induced PLD activation is reversed by the PKC inhibitor calphostin C. PKA activation does not contribute to this pathway in that the PKA inhibitor H-89 was without an effect. These findings suggest that stimulation of PLD by forskolin or isoproterenol in this tissue is not dependent on the activation of PKC. There are two known PLD isoforms (Millar et al., 1999), and it is not known whether differential activation of PLD isoforms occurs after the activation of PKC or PKA.

In the presence of forskolin or isoproterenol, the rapid activation of PLD by ACh was eliminated. The inhibition of ACh-stimulated PLD activity is PKA dependent in that the pretreatment of muscle strips with a PKA inhibitor prevented the inhibition. In these experiments, isoproterenol and forskolin were added simultaneously with ACh, suggesting that the PKA-dependent inhibition of ACh-stimulated PLD activity by isoproterenol or forskolin must occur relatively more rapidly than PKA-dependent activation of PLD. In addition, phorbol ester-stimulated PLD activity was not altered by forskolin or isoproterenol. These findings suggest that the activation of PKA by isoproterenol or forskolin inhibits a step or steps in the transduction process for muscarinic receptor activation of PLD activity.

There are at least two possible points of interaction that can account for the effect of elevated cAMP on ACh-induced PLD (Fig. 9). First, these two pathways may interact by manipulating intracellular Ca²⁺ level. It is possible that the inhibition of agonist-induced increases in Ca²⁺ interferes with optimal activation of PKC and, hence, activation of PLD. It is known that ₂ activation relaxes smooth muscle contracted by various agonists, including ACh (Torphy et al., 1983; Koenig et al., 1989), and decreases the elevation of intracellular Ca²⁺ caused by activation of M3 receptor in tracheal smooth muscle by interfering with normal Ca²⁺ cycling (Nuttle and Farley, 1996). Second, cAMP agonists have also been shown to inhibit muscarinic agonist-induced IP₃ production through a PKA-dependent mechanism (Ding et al., 1997). Moreover, it has been shown that an agonist-induced rise in IP₃ productions in bovine tracheal smooth muscle occurs at 5 to 15 s (Chilvers et al., 1989; Challiss et al., 1990). In our experiments, at 15 s, both forskolin and isoproterenol significantly inhibited ACh-induced IP₃ production (Fig. 7). This finding strongly implies a pivotal role of PLC in the regulation of ACh-induced PLD activation by cAMP agonists. It has also been demonstrated that PLC inhibition by elevated cAMP is rapid and reversible by PKA inhibitors (Fisher, 1995). Based on these observations, we speculated that modulation of ACh-induced PLD activity in tracheal smooth muscle cells by forskolin or isoproterenol is due to the regulation of PLC by PKA. Liu and Simon (1996) have shown that cAMP-dependent PKA specifically inhibits G-activated PLC-₂ isoform in COS-7 cells. They also demonstrated that PKA inhibits G-activated PLC-₂, leading to inhibition of IP₃ production in HL-60 cells. Moreover, in both cell lines, PKA rapidly and directly phosphorylates PLC-₂ in the presence of forskolin. We confirm a similar mechanism in tracheal smooth muscle cells. We have demonstrated that forskolin can phosphorylate PLC-₂ in tracheal smooth muscle cells (Fig. 8). Thus, it is probable that in tracheal smooth muscle cells, forskolin- and isoproterenol-induced increases in intracellular cAMP activate PKA, which rapidly phosphorylates PLC-₂, inhibiting IP₃ and, by implication, diacylglycerol (DAG) formation. This limits the activation of PKC and the subsequent activation of PLD. These data also suggest that the relaxation of ACh-induced airway smooth muscle contraction by ₂-adrenoceptor activation is in part due to inhibition of PLC-₂, an early transduction step in the initiation of a contractile response.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Model for effect of increased cAMP on ACh-induced PLD activation. Interaction of ACh with muscarinic receptor (M3) activates PLC, generating IP3 and DAG. IP3 mobilizes Ca2+, which can activate PKC. DAG also activates PKC. The activation of 2-adrenoreceptors stimulates adenylyl cyclase (AC), which subsequently activates PKA via cAMP. PKA in turn can inhibit the activation of PLC and, hence, reduce DAG and IP3 formation. Activation of PKA thus limits the activation of PLD and release of Ca2+ from the sarcoplasmic reticulum.

PKA has been shown to phosphorylate ₂-adrenoceptors, leading to concomitant attenuation in their ability to couple to G_s (Benovic et al., 1985; Bouvier et al., 1987). Phosphatidic acid formed as a consequence of PLD activation has also been shown to decrease adenylyl cyclase activity in fibroblasts (English and Taylor, 1991). Similar mechanisms should also reduce PLD activity after prolonged PKA activation in tracheal smooth muscle cells. This may explain the relatively slow rate of PLD activation after the stimulation of PKA in tracheal smooth muscle.

What is the function of the elevation of PLD activity by PKA? The activation of PLD with subsequent production of phosphatidic acid has been shown to be promitogenic in vascular smooth muscle (Taher et al., 1998). However, ₂ activation has been shown by Noveral and Grunstein (1994) and Schramm et al. (1996) to reduce the growth rate of airway smooth muscle cells in culture stimulated to proliferate with serum or leukotriene D₄. This apparent inconsistency may be resolved by noting that PKA activation is known to have many effects other than activation of PLD, such as decreasing intracellular Ca²⁺, activation of phosphatases, inhibiting PLC activation, and decreasing the formation of inositol phosphates, and so on. Moreover, elevation of cAMP levels has been shown to either inhibit or activate mitogen-activated protein kinase, depending on the cell types involved. However, the overall result of activation of ₂-adrenoreceptors decreases mitogenesis of cells whose growth rate has been stimulated by other substances. At present, a direct function for PLD activation during ₂-adrenoreceptor activation is not known. However, our findings that PKA regulates PLD activity in tracheal smooth muscle cells may be relevant for further investigation of the possible involvement of PLD in the molecular mechanism underlying cAMP agonist-induced smooth muscle relaxation or the complex interactions between multiple pathways that may be involved in long-term effects such as airway remodeling in asthma or chronic obstructive airway diseases.

In summary, we conclude from the present study that cAMP agonists forskolin and isoproterenol activate PLD in porcine tracheal smooth muscle cells and that the activation of PLD by these agents is at least in part PKA dependent. However, PMA-induced PLD activation is not affected by PKA inhibition. These combined results reveal that in tracheal smooth muscle, independent activation of either PKA or PKC signaling pathways leads to PLD activation. We also provide evidence for interaction between two distinct signal transduction pathways: cAMP/PKA and mAChR/PLC.