tracheal smooth muscle cells

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1 Br. J. Pharmacol. (1992), 17, '." Macmillan Press Ltd, 1992 Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells.p. Hall, S. Widdop, P. Townsend & K. Daykin Department of Therapeutics, University Hospital of Nottingham, Nottingham NG7 2UH Keywords: 1 [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2 soprenaline induced concentration-dependent [3H]-cyclic AMP formation wtih an EC5 of.2 gm. The response to 1 JM isoprenaline reached a maximum after 5-1 min stimulation and remained stable for periods of up to 1 h. After 1 min stimulation, 1 JAM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by C (1 nm), (apparent KA 1.9 x 19 M- ) indicating the probable involvement of a P2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 5 nm C there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]- cyclic AMP formation (EC5.7 JM, response to 1 AM PGE2 6.4 fold over basal). 3 Forskolin (1 nm- 1 JM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (1 mm). 4 The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (BMX) (.1 mm) and the type V, cyclic AMP selective, phosphodiesterase inhibitor rolipram (.1 mm) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. BMX (1-1 tm) and low concentrations of rolipram (<1OM), also potentiated the response to 1 JM isoprenaline. nhibitors of the type phosphodiesterase isoenzyme (SK&F 9412 and SK&F 94836) were without effect upon basal or isoprenaline-stimulated cyclic AMP responses in these cells. 5 Carbachol (1 nm- JM) produced concentration-dependent inhibition of the [3H]-cyclic AMP response to 1 JM isoprenaline in human cultured tracheal smooth muscle cells (C5.24 JM). Carbachol (1 JM) inhibited the [3H]-cyclic AMP response to 1 JM isoprenaline by 6%. This effect of carbachol was itself inhibited by atropine (5 nm) (KA 2.3 x 19M-') indicating the involvement of a muscarinic receptor. 6 These results show that primary cultures of human tracheal smooth muscle cells demonstrate cyclic AMP responses to direct receptor stimulation, adenylyl cyclase activation and inhibition with nonselective and type V-selective cyclic AMP phosphodiesterase isoenzyme inhibitors, and that the cyclic AMP response to isoprenaline can be inhibited by muscarinic receptor stimulation. Cyclic AMP; human tracheal smooth muscle; cell culture; forskolin; phosphodiesterase inhibitors; P2-adrenoceptors; muscarinic receptors ntroduction Agents which elevate tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) content can relax airway smooth muscle preparations (Barnes, 1986). This response is believed to be a consequence of several different intracellular effects of cyclic AMP including activation of Ca2" gated K+ channel activity (Kume et al., 1989), sequestration of intracellular calcium (Mueller & van Breeman, 1979), and alteration of the sensitivity of the contractile apparatus to changes in intracellular calcium concentrations (Silver & Stull, 1982). The mechanisms underlying control of tissue cyclic AMP levels have been investigated in a range of experimental animal airway tissues including bovine and canine trachealis (Torphy, 1988; Torphy et al., 1988; Hall et al., 1989; 199a; Harris et al., 1989). However, it has proved difficult to investigate the control of cyclic AMP levels in human airway smooth muscle due to the problem of obtaining sufficient tissue for studies to be performed. n order to examine the mechanisms underlying control of tissue cyclic AMP content in human tracheal smooth muscle, we have established primary cultures of tracheal smooth muscle cells from human trachealis muscle. ' Author for correspondence. This paper describes the basic characteristics in human cultured tracheal smooth muscle of the cyclic AMP responses to a range of agents capable of altering cyclic AMP content through receptor stimulation, phosphodiesterase inhibition and direct activation of adenylyl cyclase and G,. A preliminary account of this work has been presented to the British Pharmacological Society (Hall et al., 1992). Methods Primary culture of human tracheal smooth muscle cells Human tracheal smooth muscle cells were grown from explants of trachealis muscle obtained at post mortem performed within 8 h of death. Tissue was used from donors with no history of respiratory disease. A strip of trachealis taken from immediately above the carina and about 2 x 1 cm was dissected clear of surrounding tissue and transported to the laboratory in Dulbecco's Modified Eagles Medium (DMEM) containing penicillin G (2 u ml'), streptomycin (2 pg -') and amphotericin B (.5 fig `). The tissue was washed twice in 1 ml of DMEM containing antibiotics and anitfungal agents. Next, overlying mucosa was dissected free

2 CYCLC AMP N HUMAN TRACHEAL SMOOTH MUSCLE 423 from the tracheal smooth muscle under sterile conditions. Small (.2 x.2 cm) explants of tracheal muscle were then excised and about 15 explants placed in a 6 mm dish. After allowing explants to adhere, DMEM containing antibiotics, amphotericin B, 1% foetal calf serum (FCS) and glutamine (2 mm) was added to just cover the explants. Smooth muscle cell growth usually occurred about 7 days after placing explants in culture. Cultures were supplemented with fresh DMEM containing FCS and glutamine about every four days. When cells were approaching confluence in some parts of the dish, explants were removed, and 24 h later cells harvested by trypsinization. Cells from an individual dish were then plated in one 75 cm flask and grown to confluence. When confluent, each flask was split 1:4. Antibiotics and amphotericin were not added to the medium used for all subsequent passages after this stage (passage 2). Cells for experiments measuring cyclic AMP accumulation were plated in 24 ( x 1 cm) well plates and studied when confluent. Preparation of acutely dissociated tracheal smooth muscle cells Acutely dissociated tracheal smooth muscle cells were prepared by a modification of the method previously described by Kotlikoff (199). A strip of trachealis, dissected as described above, was coarsely minced with scissors and the resulting tissue fragments transferred to a Universal containing 1 ml of DMEM (without FCS) and collagenase type (Sigma) (1 mg ml-'). These were then incubated at 37C for 1 h with frequent agitation. The resultant suspension was filtered through gauze (mesh), mixed 5/5 with 1% FCS to inactivate collagenase and pelleted by centrifugation at 15 r.p.m. for 5 min. Cells were resuspended in DMEM containing 1% FCS, glutamine, antibiotics and antifungal agents then plated as described above. mmunocytochemistry All primary cell lines from each donor were examined by use of anti smooth muscle actin antibody (1:1 dilution) (Sigma) to confirm the presence of smooth muscle type cells by standard immunocytochemical techniques. Primary cell cultures used for the experiments described in this paper showed >95% of cells staining for smooth muscle actin. Accumulation of [3H]-cyclic AMP Accumulation of [3H]-cyclic AMP was measured by a modification of a previously described method (Ruck et al., 1991). n brief, confluent monolayers of cells plated in 24 well plates were labelled with [3H]-adenine (2 pci/well) for 2 h in DMEM at 37 C in an incubator constantly gassed with air /C2 (5%). At the end of this period cells were washed three times with 1 ml of Hanks/HEPES buffer, and then allowed to rewarm to 37 C for 1 min in the presence or absence of antagonists and/or phosphodiesterase inhibitors. At the end of this period, agonists were finally added for 1min before reactions were terminated by the addition of 5 fil of concentrated HCL. Cells were then stored at - 2'C. [3H]-cyclic AMP was determined following rethawing by column chromatography as previously described (Donaldson et al., 1988; Hall et al., 1989). Aliquots of ['4C]-cyclic AMP were added to each sample and the counts obtained from this recovery marker used to correct for variations in recovery from each column. n addition, a 1 pl aliquot was taken from each well of the plate after stopping reactions and counted for tritium in order to correct for variations in the number of cells per well. Statistics The affinity constant (KA) for C and atropine was determined from shifts in the agonist concentration-response curve by use of the relationship: KA = (K2/Kl)/A where A is the concentration of antagonists, K, is the concentration of agonist producing half maximal response and K2 is the concentration of agonist producing a half maximal response in the presence of antagonist. Dose-response curves were drawn with the curve fitting programme Graphpad. Statistical analysis of data was performed by paired or unpaired t tests as appropriate. All values in the text represent mean ± s.e.mean of n separate experiments. Cells from 5 individual primary tissue preparations from 3 individuals were used. Materials DMEM and FCS were obtained from Northumbria Biologicals Ltd. Plasticware was obtained from Falcon and Costar. 8-['4C]-cyclic AMP (specific activity 42.4 ptci mmol-1) and 2,8-[3H]-adenine (specific activity 26 pci mmol-') were obtained from Amersham. Gifts of C (erythro-dl- 1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol; C) SK&F 9412 (5-(4-acetamidophenyl)pyrazin-2-[1H]-one), SK&F (2-cyano-l-methyl-3-[4-(4-methyl-oxo-1,4,5,6 tetrahydropyridazine-3-yl)phenyl]quinidine; Smith Kline & Beecham, England) and rolipram (Schering AG, Germany) are gratefully acknowledged. All other chemicals were obtained from Sigma. The antibodies used for immunocytochemistry were anti smooth muscle actin (Sigma A2547) and mouse gg whole molecule (host goat) (Sigma F257). Results Effect of isoprenaline soprenaline (1 im) produced accumulation of [3H]-cyclic AMP in human cultured tracheal smooth muscle cells, the response reaching maximum after between 5 and 1min of stimulation and remaining stable for periods of up to 1 h thereafter (Figure 1). The stimulation observed with 1 gm isoprenaline was 9.5 ± 1.3 fold over basal (unstimulated) (n = 47). All subsequent experiments were performed with a 1 min stimulation period. The concentration-response curve for [3H]-cyclic AMP accumulation by isoprenaline is shown in Figure 2, and the inhibition of the response to a range of concentrations of isoprenaline by the p2-selective antagonist, C E cl < 1 -._ A 5-1 ri V n C1 C Time (min) Figure Time course of the [3H]-cyclic AMP response to 1Op1M isoprenaline in human cultured tracheal smooth muscle cells. Each point represents the mean ( ± s.e.mean, vertical bars) of triplicate determinations. The basal (unstimulated) accumulation of [3H]-cyclic AMP at time zero (Cl) and after 6 min (C2) are also shown. Data are from a representative experiment which was repeated three times with similar results on cells from a range of different passage numbers between 4 and 1. Where error bars are not shown they lie within data points.

3 424.P. HALL et al. 1 ( o- 8- CL 2 5C.2 E _ *- i Y 2- c log [soprenaline] (M) Figure 2 The effect of varying concentrations of isoprenaline on formation of [3H]-cyclic AMP in human cultured tracheal smooth muscle cells. The basal (unstimulated) level of [3H]-cyclic AMP is shown by the bar marked C, and the response to different concentrations of isoprenaline by the filled circles. Each point represents the mean ( ± s.e.mean, vertical bars) of triplicate determinations in an experiment performed in a single plate of cells. The experiment was performed 15 times with similar results < C" O- C log [so] (M) Figure 3 The effect of the P2-adrenoceptor-selective antagonist C (1 nm) () on [3H]-cyclic AMP formation in response to varying concentrations of isoprenaline (). The basal (unstimulated) response is shown in the column marked C. Each point represents the mean ( ± s.e.mean, vertical bars) of triplicate determinations in a single experiment. The experiment was repeated five times with similar results. (1 nm) (Bilski et al., 1983) in Figure 3. The calculated EC-% for isoprenaline was 2. ±.3 x O-7 M (n = 16), and the apparent KA value for C (1 nm) was 1.9 ±.3 x 19 M- (n = 6), indicating the probable involvement of the p2-receptor subtype in this response in cultured human tracheal smooth muscle cells. However, in contrast to the parallel shift in the concentration-response curve produced by lnm C when lnm to 1lM isoprenaline was used as agonist (Figure 3), in experiments examining the effect of C (5 nm) upon the response to the higher concentrations of isoprenaline used (>1M), C produced a nonparallel shift of the concentrationresponse curve to isoprenaline, with a reduction in the maximum response to isoprenaline (Figure 4). Effect ofpassage number on isoprenaline response n order to define the stability of the cyclic AMP response in cells from different passage numbers, the fold stimulation and EC5 for isoprenaline induced [3H]-cyclic AMP formation was compared between cells from passage 4 and 12. No significant change was seen with increasing passage number during this period. However, after passage 12 some decline in the maximum stimulation by isoprenaline was observed. All subsequent experiments were therefore performed on cells C log [so] (M) Figure 4 The effect of the P2-adrenoceptor-selective antagonist C (5 nm) () upon [3H]-cyclic AMP formation in response to isoprenaline (). The basal (unstimulated) response is shown by the column marked C. Each point represents the mean (± s.e.mean, vertical bars) of triplicate determinations in a single experiment. The experiment was repeated three times with similar results. from passages between 4 and 12. The effect of passage number upon the responses to prostaglandin E2 (PGE2), forskolin and rolipram were also examined. The potency of these agents and the stimulation observed with these agents remained stable between passages 4 and 12. Response to isoprenaline in collagenase-treated cell preparations n order to compare the response of cells after several passages with cells maintained in short term culture, experiments were performed using cells acutely dissociated with collagenase and plated in 24 well plates to examine the responses to isoprenaline. From a single tissue preparation, inadequate cell numbers were obtained to perform full concentrationresponse curves to agonists. Therefore, cells obtained by primary collagenase digestion were grown to confluence in 24 well plates and responses to isoprenaline examined (i.e. passage 2). The EC5 for isoprenaline-induced cyclic [3H]-cyclic AMP formation was 9 ± 1 x 1-7 M (n = 4), comparing well with the responses seen in cells after repeated passage. Effect ofprostaglandin E2 Prostaglandin E2 (PGE2) also stimulated concentrationdependent [3H]-cyclic AMP formation in primary cultures of human tracheal smooth muscle cells (Figure 5). The EC5 for 12 - C log [PGE21 (M) Figure 5 The effect of varying concentrations of prostaglandin E2 (PGE2) on [3H]-cyclic AMP formation in human cultured tracheal smooth muscle cells. Data are shown from a single representative experiment which was repeated four times with similar results. Each point represents the mean ( ± s.e.mean, vertical bars) of triplicate determinations. The control response is shown by the column marked C.

4 CYCLC AMP N HUMAN TRACHEAL SMOOTH MUSCLE 425 this response to PGE2 was 1.1 ±.2 X 1OM (n = 5). The response to 1 ALM PGE2 was 6.4 ±.5 fold over basal (unstimulated) (n = 5). Effect offorskolin The adenylyl cyclase activator, forskolin, produced concentration-related [3H]-cyclic AMP formation in human cultured tracheal smooth muscle cells as shown in Figure 6. This response was non-maximal with the concentrations of forskolin used (1 nm to 1 M). The response to 1 EM forskolin was increased in the presence of the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl- 1- methylxanthine (BMX) (.1 mm) (Figure 6). The direct acting G protein stimulant, NaF (1O mm), also elevated cell [3H]-cyclic AMP content to 1.6 ±.3 fold over basal (P<.5, n=4). Effect ofphosphodiesterase inhibitors The effect of the nonselective PDE inhibitor, BMX, on both basal and isoprenaline-stimulated cyclic AMP responses is shown in Figure 7. t can be seen that alone, BMX produced a small but significant elevation of tissue cyclic AMP content. n addition, the response to isoprenaline (1 AM) was also increased in the presence of BMX (1ZM-1 mm) (Figure 7, Table 1). n order to attempt to define the PDE isoenzymes important for the physiological control of whole cell cyclic AMP content, the effects of both type and type V PDE inhibitors were examined. The data obtained from these experiments with the type selective inhibitors, SK&F 9412 and SK&F 94836, and the type V selective inhibitor, rolipram, are shown in Table 2. t can be seen that whilst inhibitors of the type isoenzyme produced no significant effect on basal cyclic AMP responses, rolipram, an inhibitor of the high Km, cyclic AMP hydrolysing isoenzyme present in tracheal smooth muscle (Nicholson et al., 199) produced significant elevation of cyclic AMP levels (Table 1). Low concentrations of rolipram (< 1 AM) in the presence of 1 LM isoprenaline produced a small concentration-related increase in cyclic AMP levels (response to 1 AM rolipram 1.7 ±.2 fold over response to 1 AM isoprenaline alone, n = 1, P<.5; EC5.3 ±.1 AM, n = 6). However, high concentrations of rolipram (>1AM) had no significant effect upon the response to 1 AM isoprenaline (Table 2). 6 E 6. 4' CL. 2 C +F log [Forskoli.n] (M) Figure 6 The effect of varying concentrations of forskolin (@) on [3H]-cyclic AMP formation in human cultured tracheal smooth muscle cells. Also shown are the basal (unstimulated) response in these cells (C), the response to 3-isobutyl-l-methylxanthine (BMX) (.1 mm) () and the response to forskolin (1 1M) and BMX (.1 mm) together ( + F). Each point represents the mean (± s.e.mean, vertical bars) of triplicate determinations. The experiment was repeated twice with similar results E 6. 1 a-.2 L) 5 * -5-4 log [BMX] (M) Figure 7 The effect of varying concentrations of 3-isobutyl-1- methylxanthine (BMX) on [3H]-cyclic AMP formation in human cultured tracheal smooth muscle cells in the absence (x) and presence () of FM isoprenaline. The basal (unstimulated) level, and the response to 1 ;AM isoprenaline alone are shown in the columns marked C and so respectively. Each point represents the mean ( ± s.e.mean, vertical bars) of triplicate determinations in a single plate of cells. The experiment was repeated three times with similar results. The mean changes in [3H]-cyclic AMP levels with these agents are summarized in Table 2. Agent Table 1 Effect of a range of agents on [3H]-cyclic AMP formation in human tracheal smooth muscle cells soprenaline (1 JAM) Forskolin ( 1AM) Prostaglandin E2 (1 AM) NaF (1mM) -3 Fold stimulation over control - BMX (n) + BMX (.1 mm) (n) 9.5 ± 1.3 (47) 2.±.3 (5) 6.4 ±.5 (5) 1.6±.3 (4) 28.5 ± 1.9 (9) 4.4 ±.4 (3) ND ND The responses to all agents were significantly (P <.5) larger than unstimulated values for [3H]-cyclic AMP accumulation. ND = not determined. All responses are given as the mean fold stimulation over basal in n separate experiments 3-sobutyl-l-methylxanthine (BMX) (.1 mm) alone produced a 1.8 fold increase over basal. Effects of carbachol upon isoprenaline-stimulated cyclic AMP responses n order to examine for the possibility of the involvement of muscarinic receptor stimulation in inhibiting adenylyl cyclase activity in human cultured tracheal smooth muscle cells, the effect of the muscarinic agonist, carbachol, on isoprenalinestimulated cyclic AMP formation was examined (Figure 8). Carbachol produced concentration-dependent inhibition of the [3H]-cyclic AMP response to 1 1AM isoprenaline, with an C5 value of 2.4 ±.2 x 7- M (n = 6). Carbachol (1 1AM) inhibited the response to 1 1AM isoprenaline by 6± 1% (n = 8, P<.1). The effect of carbachol was itself inhibited by atropine (5 nm) (KA 2.3 ±.5 x 19 M-1, n = 4), indicating the involvement of a muscarinic receptor in this response (Figure 9). Discussion n this paper, we describe the characteristics of the cyclic AMP responses to a range of agents in primary cultures of human tracheal smooth muscle grown from tissue explants. Elevation of cellular cyclic AMP content in primary cultures of human tracheal smooth muscle cells was observed following stimulation with isoprenaline, prostaglandin E2, inhibition of phosphodiesterase activity by nonselective and type

5 426.P. HALL et al. Table 2 Effect of phosphodiesterase inhibitors on [3H]-cyclic AMP responses in human tracheal smooth muscle cells Agent BMX (.1 mm) Rolipram (.1 mm) SK&F 9412 (.1 mm) SK&F (.1 mm) Fold stimulation over control - soprenaline (n) + soprenaline (1 gm) (n) 1.8 ±.3* (9) 1.5 ±.2* (9) 1. ±.1 (4) 1. ±.2 (8) 2.1 ±.2* (9) 1. ±.1 (9).8±.1 (4).9±.1 (5) *P <.5 compared with control. The response to 1 LM isoprenaline alone was 9.5 ± 1.3 fold over control. The responses shown in the presence of isoprenaline are the fold stimulation compared with the response to glm isoprenaline alone. Responses shown are the mean ( ± s.e.mean) values relative to either unstimulated or 1 gm isoprenaline-stimulated cells obtained from n separate experiments. BMX = 3-isobutyl-1-methylxanthine. E Q 5( C CCh so -8-7 log [CCh] (M) -5 Figure 8 The inhibition of the [3H-cyclic AMP response to 1 ZtM isoprenaline by varying concentrations of carbachol (CCh) (). The control response to LM isoprenaline is shown by the column marked so and the basal (unstimulated) level by the column marked C. Each point represents the mean (± s.e.mean, vertical bars) of triplicate determinations in a single experiment which was repeated three times with similar results. The effect of mm carbachol alone on [3H]-cyclic AMP formation is shown in the column marked CCh. E ~ 1-5- : C so -8-7 log [CCh] (M) -5-4 Figure 9 The effect of atropine (5 nm) () on the inhibition of the [3H]-cyclic AMP response to jtm isoprenaline by varying concentrations of carbachol (CCh) (). The control response to isoprenaline (1 gtm) is shown by the column marked so and the basal response by the column marked C. Each point represents the mean ( ± s.e.mean, vertical bars) of triplicate determinations in a single experiment which was repeated three times with similar results. V (cyclic AMP)-selective phosphodiesterase inhibitors, and by direct stimulation of adenylyl cyclase or G, by forskolin and NaF respectively. Both prostaglandin E2 and isoprenaline induced concentration-related cyclic AMP formation in human cultured tracheal smooth muscle. The response to isoprenaline was inhibited by the P2-adrenoceptorselective antagonist, C (Bilski et al., 1983), indicating the probable involvement of the p2-receptor subtype in this response. The observed KA value of 1.4 x 19 M -, is similar to the value previously reported for inhibition of p2-responses in guinea-pig atria (Bilski et al., 1983), and for the P2-receptor-mediated inhibition of histamine-stimulated inositol phosphate formation in bovine tracheal smooth muscle slices (Hall & Hill, 1988), but approximately 1 fold higher than the value for inhibition of P-receptor responses in uterus (Bilski et al., 1983). However, when concentrations of isoprenaline producing maximal cyclic AMP responses were used, 5 nm C produced a than would have been expected greater degree of inhibition from the data obtained with 1 nm C using lower concentrations of isoprenaline as agonist. The explanation for this observation is not clear, although one possibility is that with the higher concentration of C , the antagonist may be behaving in a noncompetitive manner. This possibility would be worthy of further study in other cell lines expressing P2-adrenoceptors. The adenylyl cyclase activator forskolin, as would be expected, also induced [3H]-cyclic AMP formation, the response being nonmaximal over the concentration-range examined (1O nm- 1 LM). n addition, NaF also produced elevation of cell cyclic AMP content, presumably through activation of G, These data are in keeping with previous observations in bovine tracheal smooth muscle, where NaF produced significant elevation of cyclic AMP levels (Hall et al., 199a). n this latter study, NaF also inhibited forskolininduced cyclic AMP formation, presumably by a direct action on GQ. A range of phosphodiesterase isoenzymes are present in smooth muscle homogenates from both human (Torphy & Undem, 1991) and animal (Torphy & Cieslinski, 199; Nicholson et al., 1991; Shahid et al., 1991) airways. We have previously shown that in bovine trachealis muscle, the type V enzyme is important in controlling tone (Hall et al., 199b,c) and cyclic AMP content (Hall et al., 1989). Rolipram is an effective and potent inhibitor of the type V isoenzyme in homogenates from this tissue (Nicholson et al., 199). The role for the type isoenzyme remains less clear. nhibitors of the type isoenzyme have been shown to produce small degrees of relaxation of precontracted airway smooth muscle preparations from a range of species at concentrations similar to those used in the present study, although these agents do not necessarily elevate whole tissue cyclic AMP content (Gristwood et al., 1986; Silver et al., 1988; Torphy et al., 1988; Hall et al., 1989; 199b). This may be due to a degree of compartmentalisation within the cell of phosphodiesterase isoenzymes and/or pools of cyclic AMP. The results obtained in the present study are consistent with this suggestion. We observed increases in both basal and isoprenaline-induced cyclic AMP responses in the presence of the nonselective phosphodiesterase inhibitor, BMX. n keeping with previous observations, the absolute rise in cyclic

6 CYCLC AMP N HUMAN TRACHEAL SMOOTH MUSCLE 427 AMP levels seen with BMX in the absence of another agent to stimulate adenylyl cyclase was small, suggesting that the basal level of cyclic AMP production in tracheal smooth muscle cells is low. Rolipram, an inhibitor of the high Km, cyclic AMP hydrolysing isoenzyme present in human airway smooth muscle (Torphy & Undem, 1991) and a potent bronchodilator in vivo (Harris et al., 1989) elevated basal cyclic AMP levels in human cultured tracheal smooth muscle cells, and at concentrations selective for the type V isoenzyme produced a small potentiation of the response to 1 gm isoprenaline with an EC_% of.3 SM, similar to that reported in other animal studies (Shahid et al., 1991; Torphy & Undem, 1991). However, high (>1OM) concentrations of rolipram had no significant effect upon isoprenaline-induced cyclic AMP formation. The type (low Km cyclic AMP hydrolysing) phosphodiesterase isoenzyme inhibitors, SK&F 9412 and SK&F 94836, (Reeves et al., 1987; Torphy et al., 1988) were without significant effect upon basal cyclic AMP levels. The effects of these agents upon basal levels of cyclic AMP accumulation are similar to those previously reported in bovine trachealis (Hall et al., 1989). The lack of effect of high (as opposed to low) concentrations of rolipram upon isoprenaline-stimulated cyclic AMP formation does, however, remain to be explained. n some cell types, muscarinic receptor stimulation inhibits adenylyl cyclase activity through stimulation of M2 or M4 receptor subtypes. n the present study, marked reduction of the cyclic AMP response to isoprenaline was observed in the presence of the muscarinic agonist, carbachol. These results are in agreement with previous observations in canine trachealis (Jones et al., 1987; Torphy et al., 1987; Sankary et al., 1988) but in contrast to findings in whole tissue from bovine trachealis (Hall et al., 199c). The results in the study described in this paper would argue for the presence of an inhibitory effect of muscarinic receptor stimulation on cyclic AMP levels in human airway smooth muscle. A similar inhibitory effect of carbachol on isoprenaline-induced cyclic AMP formation was observed in a recent study in canine tracheal smooth muscle cells (Yang et al., 1991). n primary cultures of human tracheal smooth muscle, cyclic AMP responses to isoprenaline, forskolin and phos- *phodiesterase inhibitors remained stable over a period of at least 8 cell passages, demonstrating that primary cultures of human tracheal smooth muscle cells provide a potentially useful model system for the study of the regulation of airway cyclic AMP responses. The response to isoprenaline in cells from passages 4 to 12 was similar to the response seen in acutely dissociated cells grown to confluence in short term cultures. t was also hoped to compare the response in cultured cells with the response in acutely dissociated cells ex vivo but inadequate cell numbers precluded accurate estimation of concentration-response curves in the acutely dissociated cell preparations. Even if this were possible, however, collagenase treatment could potentially affect cell surface receptor number. Further evidence to suggest that coupling of the P-adrenoceptor to adenylyl cyclase in cultured cells is similar to coupling in vivo is provided from the observation that the EC5 value for isoprenaline induced cyclic AMP formation in the cultured cells compares well with the value obtained for this response in previous studies of canine trachealis muscle (about.21im, Fujiwara et al., 1988). The concentration-response curve for isoprenaline-induced cyclic AMP formation in primary cultures of human tracheal smooth muscle cells, and in previous studies of canine tracheal smooth muscle ex vivo, both lie approximately one log point to the right of the concentration-response curve for relaxation of human airway preparations (de Jongste et al., 1989). This would imply that a significant spare receptor reserve exists for P2-adrenoceptor-induced cyclic AMP formation in human airway smooth muscle. n conclusion, this study describes the characteristics of the cyclic AMP responses to isoprenaline, prostaglandin E2, forskolin, NaF, nonselective phosphodiesterase inhibition and a range of phosphodiesterase inhibitors in primary cultures of human tracheal smooth muscle cells. These responses are quantitatively and qualitatively similar to those previously described in ex vivo airway smooth muscle preparations from other species. 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