185
J Physiology (1992) 86, 185-194
© Elsevier, Paris
Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture N Marmy, W Durand-Arczynska, J Durand* lnstitut de Physiologie, 5, rue du Musde, CH-1700 Fribourg, Switzerland
(Received 2 July 1992; accepted 20 August)
Summary - Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against m-smooth muscle actin (~SMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[3H]inositol in order to label the phosphoinositide pool, and the various [3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IPi isomers were separated in three peaks; IP2, IP3, IP4, IP5 and IP6 (phytic acid) were each eluted as single peaks. The identity of the [3H]peaks was verified with corresponding [3H]IP standards. The accumulation of [3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCI, with or without a bronchoconstrictor agent (carbachol, histamine, PGF2c0. Histamine, 10-4 M, elicited a four times larger IP accumulation than carbachol, 10-~ M, and than PGF2c, 5 10-5 M. Dose-response curves were established for histamine and carbachol in the range 10-7-10-4 M. At 10-7 M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways. inositol phosphates / airway smooth muscle / histamine / carbachol / prostaglandin F2,~
Introduction The c o n t r a c t i o n of airway smooth muscle is controlled by n u m e r o u s b r o n c h o - a c t i v e mediators that are released from various sources, principally the a u t o n o m i c n e r v o u s system, the n o n - a d r e n e r g i c n o n - c h o l i n e r g i c ( N A N C ) system and paracrine actions from various cell types (Barnes et al, 1991; Forrest and Lee, 1991; L a i t i n e n and Laitinen, 1991; White and Kaliner, 1991; R o b i n s o n and Holgate, 1991). A l t h o u g h a wealth of data has b e e n gathered regarding physiological, pharmacological and b i o c h e m i c a l aspects of the control of airway caliber, little i n f o r m a t i o n is available on the m e c h a n i s m of response of h u m a n b r o n c h i to constrictor agonists at the cell level. The detailed study of post-receptor events leading to contraction in h u m a n airway SMC has been h a m p e r e d by the limited d e v e l o p m e n t of a cell preparation * Correspondence and reprints.
suitable for b i o c h e m i c a l analysis of the m o l e c u l a r c o m p o n e n t s of b r o n c h o c o n s t r i c t i o n . In recent years, a cell culture model was reported that retained p h e n o t y p i c characteristics closely resembling those of the native tissue (Twort and van Breemen, 1988). It is generally agreed that, in the airways, the principal process in the coupling between the extracellular signal and the increase in cytosolic Ca 2+ c o n c e n t r a t i o n which takes place during smooth muscle activation, involves the stimulation of phospholipase C (PLC) that catalyses the b r e a k d o w n of p h o s p h a t i d y l i n o s i t o l 4,5bisphosphate, thus generating two second messengers, 1,4,5-IP3 and diacylglycerol. The latter is k n o w n to activate protein kinase C, while 1,4,5-IP3 triggers the rapid release of Ca 2+ from intracellular stores (Berridge and Irvine, 1989). The possible occurrence of Ca 2+ influx through receptor-operated or voltage-sensitive m e m b r a n e c h a n n e l s is not clearly established in the airways.
186 The aim of the present study was to characterize the f u n c t i o n a l properties of h u m a n bronchial SMC in culture by looking at the pattern and at the time course of the a c c u m u l a t i o n of IPs in these cells. For that purpose, the action of several b r o n c h o c o n s t r i c t o r agents was e x a m i n e d on the IP turnover. H i s t a m i n e was used as a test substance because of the i m p o r t a n c e of mast cells in the pulmonary tissue and in view of the characteristic contractile response to this agent in m a m m a l i a n , i n c l u d i n g h u m a n , lung. Carbachol was chosen because the parasympathetic nervous system is the d o m i n a n t neural b r o n c h o c o n s t r i c t o r m e c h a n i s m , playing an important role in the regulation of airway tone. One of the major prostanoids synthetized in the bronchi is p r o s t a g l a n d i n F2c~ (PGF2cd which is able to modulate directly the airway tone by a paracrine or autocrine mode. The physiological significance of this study deals with the res p o n s i v e n e s s of intact cells from h u m a n airways at the m e m b r a n e level. In order to e x a m i n e the first post-receptor events upon agonist stimulation, b i o c h e m i c a l techniques were chosen to measure the IP turnover. It was thus possible to assess the i n v o l v e m e n t of PLC in the m e c h a n i s m of m e m b r a n e t r a n s d u c t i o n operated by agents k n o w n to elicit b r o n c h o c o n s t r i c t i o n in vivo. The results showed that the m e m b r a n e receptors for the agonists, as well as the first post-receptor events and also the f u n d a m e n t a l features of IP m e t a b o l i s m were retained in these cultured cells and that the chosen agonists were effective in accelerating the IP turnover. No previous report was so far available c o n c e r n i n g the production of IPs in h u m a n airways in response to b r o n c h o c o n strictor agents. This preparation seems to be a suitable model to study the m e c h a n i s m s i n v o l v e d in the control of airway smooth muscle contraction. Parl of the results were published in abstract form ( M a r m y et al, 1992).
Materials and m e t h o d s Cell isolation a n d c u l t u r e
Human bronchi were obtained from patients undergoing surgery for carcinoma of the lung. Fragments of noncarcinomatous bronchi were removed within 5 min of resection and placed into a modified Krebs-Ringer-Henseleit solution (KRH) containing (in mM): 116.8 NaCI, 25 NaHCO3, 5.9 KCI, 1.2 Nail2 PO4, 5.5 glucose, equilibrated at pH 7.4 in an atmosphere of 5% CO2 in air. 15-20 min following sampling, cells were isolated
according to the procedure of Twort and van Breemen (1988). The smooth muscle was dissected under sterile conditions, cleaned of the thin epithelial layer and connective tissue and placed in KRH supplemented with penicillin 100 U/ml, streptomycin 100 Bg/ml and garamycin 2 gg/ml. The tissue was then minced into thin pieces and incubated 2 h in 2 ml of KRH containing 1 mg/ml collagenase (type IA), 15 U/ml elastase, 0.5 mg/ml DNase (type IV), 1 mg/ml dithiothreitol and 1% bovine serum albumin. The incubation was per formed at 37"C in a shaking water bath in an atmosphere of 5% CO2 in air. The reaction was terminated by the addition of 5 ml culture medium which was a mixture of Ham's El2 and Dulbecco's modified Eagles (DMEM) culture medium (1:1) containing 10% fetal calf serum (FCS), 1~ non essential aminoacids, 100U/ml penicillin, 100Bg/ml streptomycin, 5 Bg/ml insulin and 2.5 mM glutamine. The tissue was dispersed by mild pipetting and passed through a gauze filter. The cell suspension was centrifuged and the pellet was diluted with culture medium. The cells were seeded into a 35-mm diameter plastic dish; they were grown at 37°C in a water-saturated atmosphere containing 5% CO2 in air. After 2 h of incubation, the medium was cautiously removed and replaced with fresh medium, thus eliminating cells remaining in suspension after this period. Amphothericin B (28 gg/ml) was added to the medium during the first 20 h. The culture medium was replaced after 20 h and every 48 h thereafter. Cells at confluence were subcultured, using 1 ml of a non-enzymatic cell dissociation solution supplemented with 0.25% trypsin. Alternatively, aliquots of cells were frozen and stored in liquid N2 in culture medium containing 40% FCS and 1074 DMSO. lnzmunocytochemistry
The cells were identified in subcultures by using a monoclonal antibody against c~SMA which has been shown to be a specific marker of smooth muscle (Skalli et al. 1987). Cells were grown for 3 days in medium containing 10% FCS and then 5 days in serum-free medium, in order to stop cellular proliferation, thus increasing the expression of o~SMA (Owens et al, 1986; Blank et al, 1988). The dishes were rinsed five times with a balanced salt solution (BSS; NaC1, 102 mM, NaHCO3, 20mM, KCI, 6mM, NaH2PO4, 1.2raM, Hepes, 24 mM, pH 7.4), then the cells were fixed for 30 min in Bouin solution containing: 75% saturated picric acid solution, 20% formaldehyde and 5% glacial acetic acid. The dishes were rinsed again and the membranes of the cells were permeabilized by incubation with 0.4% Triton X-100 in BSS during 4 h at 20"C on a shaking plate. Then, the dishes were washed with BSS and the non-specific binding sites for peroxidase were blocked by a 30-min incubation with 3% goat serum and 5% albumin in BSS. The antibody against ~SMA (primary antibody) was then added at a dilution of 1:50¢)
187 in BSS and the dishes were gently shaken overnight at 4°C. On the next day they were washed with BSS and incubated for 3 h with the secondary antibody, capable of binding to the primary and conjugated to biotin, at a dilution of 1:200. After washing, the cells were incubated for 2 h with a complex of peroxidase conjugated to avidin and biotin, capable of binding with high affinity to the secondary biotinylated antibody. After a final washing, substrates for peroxidase (DAB and H2 02 ) were added and a braun staining (DAB oxidation) of the o~SMA filaments appeared after 1 or 2 rain. The nuclei were then counterstained with the hematoxylin reagent.
1988). The [~H]IPs were separated, according to the following protocol: after 10rain migration with water during which myo@H]inositol was eluted, a gradient from 0-0.9 M buffer was applied over 38 rain; the latter concentration was maintained from the 48th to the 55th min. Radioactivity was monitored on-line with a FIoone detector with low level counting capacity (A250x, Canberra Packard, Ztirich, Switzerland) using the scintillant Flo-Scint IV (Canberra Packard) in a ratio of 4:1, scintillant to eluent.
Production o f radiolabelled IPs
The DNA content of cells was measured according to the method of Labarca and Paigen (1980). The cells in culture dishes (three dishes for each experiment) were thoroughly scrapped, mixed, then sonicated 15 s (Brunk et al, 1979) in l ml phosphate-saline buffer (8 mM NaH2PO4, 40raM Na2HPO4, 2.0M NaCI, 2raM EDTA, pH 7.4). The fluorescence intensity of the complex that DNA forms with the compound Hoechst 33258 was measured with a spectrofluorophotometer Shimadzu RF 5000, using calf thymus DNA as standard. The DNA content of each dish was measured in triplicate determinations.
Cultured cells between passage 3 and 8 (Murray and Kotlikoff, 1991 ) were used for studies of IP production. The cells were grown on 35-ram dishes. Three days before experiments the culture medium was removed and replaced with 2 m l fresh medium containing 185KBq/ml (51.tCi/ml) of myo-[3H]inositol and the cells were allowed to reach confluence. For the analysis of labelled IPs, each dish was rinsed five times with l ml serum-free medium containing no radioactivity. The medium was maintained at 37°C in a water bath and pre-equilibrated with a gas mixture containing 5% CO2 in order to avoid any thermic shock or pH perturbation. The cells were then preincubated for l0 rain with 2 ml of medium containing 10 mM LiCI in a shaking water bath at 37°C (Berridge et al, 1982; Chilvers et al. 1989; Brami et al, 1991). Following a rapid aspiration of the medium, the period of IP determination was started by adding 1 ml of the same medium containing either histamine, PGF2c~, carbachol, or no agohist. After a defined incubation period (1-30 min), the stimulation was terminated by rapid vacuum aspiration of the medium, followed immediately by addition of 600 gl 5% ice-cold trichloroacetic acid (TCA) containing 50 lag/ml phytic acid (Horstman et al, 1988). The dishes were then placed on ice during l0 min, the cells thoroughly scrapped, and the material transferred into an Eppendorf microfuge tube. The dishes were then rinsed twice with 150 gl of 5% TCA that were also transferred into the tube; the lysed cells were pelleted by centrifugation (11 300 g, 5 rain). The supernatant was collected and TCA was removed by five extractions with 2 ml of water-saturated diethyl ether (Berridge et al, 1983; Dean and Moyer, 1987; Horstman et al, 1988). The extract was then neutralized with 50 gl 0.1 N NaOH before being injected into the HPLC column.
Quantitative determination o f DNA content
Materials Collagenase type 1 A, calf thymus DNA, DNAse 1, dithiothreitol, trypsin, amphotericin B, anti aSMA, peroxidase conjugate, histamine, PGF2c~, carbacbol, atropine, diphenhydramine and non-enzymatic cell dissociation solution were purchased from Sigma (St Louis, MO, USA). Penicillin, streptomycin, L-glutamine, fetal calf serum, non-essential aminoacids, DMEM and Ham's FI2 medium were obtained from Flow Laboratories (Baar, Switzerland). Medium 199 was purchased from Gibco (Basel, Switzerland). Garamycin was obtained from Shering Corporation (Kenilworth, USA). myo-[3H]inositol and [3H]IPs standards were purchased from New England Nuclear (Du Pont de Nemours International, Ztirich, Switzerland). Other reagents (analytical grade) were from Fluka (Buchs, Switzerland), Merck (Darmstadt, Germany), or Essex (Lucerne, Switzerland).
Results Cell growth and characterization
Separation o f [3H]IPs by H P L C The [3H]IPs were separated by HPLC (Varian 9010, Basel, Switzerland) on a PRP-XI00 anion exchange column (Hamilton, Bonaduz, Switzerland) with a potassium phosphate buffer, pH 3.35, at a flow rate of 1 ml/min (Dean and Moyer, 1987; Horstman et al,
S M C from human airways, plated at a density o f 2.5 103 c e l l s / c m 2, e x h i b i t e d a lag period of 2 - 3 days, then proliferated rapidly (cell population d o u b l i n g time 1.8 day) and r each ed c o n f l u e n c e in 12-14 days. Figure 1 shows a typical growth curve. Before reaching c o n f l u e n c e , cells were
188
cells / cm 2 (x 10 4) 16 12 8 4 0
~lt
I
I
I
0
4
8
12
days Fig 1. Growth curve for subcultured human bronchial SMC. Cells were plated at a density of 2500 cells/era 2. Each point is the mean of triplicate cell counts from two dishes. Between day 4 and 8, the population doubling time was 1.8 days.
loosely arranged on the dishes and, as a distinctive feature, they did not migrate to any extent (Chamley-Campbell et al, 1979). Several nucleoli were generally present in the nucleus. The cells at confluence appeared elongated, spindle-shaped and densely packed together, building a highly organized hills-and-valleys structure, as reported also by Panettieri et al (1989). The cells were subcultured for 8-10 passages without noticeable alteration of their appearance (fig 2). Cells stored in liquid nitrogen could be thawed, then cultured over several passages without apparent alterations in growth nor in functional properties, as previously reported for human vascular SMC (Dartsch et al, 1990). SMC were identified by their positive reaction with a monoclonal antibody against c~SMA (Skalli et al, 1987). The staining revealed the presence of strands of parallel, highly ordered, filaments running through the whole cell (fig 2). The immunocytochemical characterization was performed for each cell line, at different suhpassages, as well as following thawing of cells stored in liquid nitrogen. At least 95% of the cells were positively stained; however, differences in the intensity of c~SMA labelling were noticed between individual cell lines. Contaminant epithelial cells
Fig 2. Photomicrographs of human bronchial SMC in subculture, a. Phase microscopic appearance of cells 4 days after the 6th passage, b. Microscopic appearance (without phase contrast) of subconfluent cells at the 5th passage. The cells were labelled with a monoclonal antibody against ~SMA; the actin filaments were evidenced with the immunoperoxidase technique and the nuclei counterstained with the bematoxylin reagent. Bars 50 ~tm (a) and 25 p.m (b).
189 did not survive after one passage, as observed previously (Panettieri et al, 1989). Accumulation o f IPs in subcultured cells The ability of the cultured cells to produce IPs in response to three agonists of bronchoconstriction, carbachol, PGF2c~ and histamine, was examined. Figure 3 shows three HPLC chromatograms obtained with a cocktail of selected [3H]IP standards or with materials extracted from cells cultured for 3 days in the presence of myo-[3H]inositol. Panel a shows the
1-I P 1,4-IP2 4"IP 1 1,4,5-IP3
cpm 1000
1,3,4,5-IP4
(a)
:~...
~
:
-
epm 500
IP ? (b)
?
0 , . '! I ,
.
~,~3
. . . . . . .
IP6 ? /%.,
cpm 5OO
0
i0
20
30
40
50
time (min)
3. HPLC chromatograms of [3H]IPs. a. Elution pattern of the indicated [3H]IP standards, b. Elution profile of [3H]IPs extracted from untreated cells incubated for 3 min under standard conditions, c. Elution profile of [3H]IPs extracted from cells incubated for 3 rain in the presence of 104 M histamine. The elution protocol was: 10 rain water; a gradient from 0 to 0.9 M phosphate buffer, pH 3.35, over 38 rain; 0.9 M phosphate buffer from 48 to 55 rain. Fig
elution profile of authentic [3H]IPs. The retention times were: 18min for [3H]I-IP, 19min for [3H]4-IP, 2 4 m i n for [3H]I,4-IP2, 31 min for [3H]I,4,5-IP3 and 36 rain for [3HI 1,3,4,5-IP4. For the experiment shown in panel b, cells were incubated for 3 min under standard physiological conditions (culture medium equilibrated with a gas mixture containing 5% CO2 in air, yielding a pH of 7.4 at 37°C) in the absence of agonist; myo[3H]inositol was eluted between 2 and 8 min. A peak corresponding to none of the standards was found to appear at 17 min. The followin~ peaks had retention times similar to those of ["H]I-IP, [3H]4-IP and [3H] 1,4-IP2. The next small peak to PHPear had a retention time similar to that of ]1,4,5-IP3; it was barely detectable on the representative chromatogram shown in figure 3 (b). However, it was reported that this material contains both 1,4,5-IP3 and 1,3,4-IP3 (Dean and Moyer, 1987; Murray et al, 1989). Subsequently eluted peaks represented probably [3H]IP5 and [3H]IP6; however, they were not identified with the aid of corresponding standards. The run shown in panel c was performed under identical conditions, except that histamine 10 4 M was present during the 3 min of incubation. The following observations were made: i) the unidentified [3H]IP was not altered by histamine treatment, whereas [3H]I-IP and [3H]4-IP were conspicuously enhanced; ii) [3H]l,4-IP2 was increased; iii) a sizable [3H]IP3 peak was recorded and [3H]IP4 could also be resolved; and iv) the peaks putatively corresponding to [3H]IP5 and [3H]IP6 were not influenced by histamine. The time course of [3H]IPs accumulation observed in the absence of agonist or elicited by carbachol or histamine was examined and the results are shown in figure 4. The total amount of [3H]IPs was the sum of surface areas of all peaks eluted by HPLC. Between 1 and 30 rain of incubation in the absence of agonist, the production of [3H]IPs was nearly constant. Carbachol, 10 -4 M, enhanced the total production of [3H]IPs over the time period examined; the increase was 52% at 30min. Histamine, 1 0 ~ M , caused a stronger stimulation than carbachol; the increase was roughly linear with time; it reached a value five times above the basal level at 30 rain. The linear increase due to histamine suggested that the production of radiolabelled IPs was not limited by the consumption of the radiolabelled phosphatidyl-inositol stores in the membranes, although the turnover of [3H]IPs was inhibited by LiC1.
190
A
cpm [3H] IPs / mg DNA (x 10 5) 40
30
c o u n t s o f t h e v a r i o u s 13HIlP p e a k s e l u t e d f r o m t h e H P L C c o l u m n w e r e s u m m e d up. F o r e a c h c o n centration, control values were determined with u n s t i m u l a t e d s a m p l e s r u n in p a r a l l e l w i t h c e l l s treated with agonist; these values were systematically deducted from the data obtained with agonist-treated samples. Figure 5 shows the dose-reponse curves for carbachol and histamine.
cpm [3H] IPs / mg DNA (x 10 5) 0
10
20
4
30
cpm [3H] IPs / mg DNA (x 10 5)
A
B
2
120 /I/IF
i
I
I
|
7 6 5 4 - log [carbachol]
80 40 0
I
0
10
I
20
I
cpm [3H] IPs / mg DNA (x 10 5) 20
B
30
time (min) Fig 4. Time course of total [3H]IPs accumulation. Cells were incubated in the presence (solid circles: carbachol (A), solid squares: histamine (B)) or in the absence (open circles) ol 10 4 M agonist for the corresponding period. Each point is the mean of two experiments.
Characterization of the responsiveness of the cells to constrictor agents To f u r t h e r c h a r a c t e r i z e t h e r e s p o n s i v e n e s s o f the c e l l s , t h e p o t e n c y o f c a r b a c h o l a n d h i s t a m i n e to e l i c i t t h e f o r m a t i o n o f [ 3 H ] I P s in t h e s e c e l l s w a s also examined, based on dose-response relationships. For these experiments, a single incubation t i m e o f 3 rain w a s c h o s e n a n d t h e r a d i o a c t i v i t y
10
0
// 7 6 5 - log [histamine]
4
Fig 5. Dose-response curves of [3H]IP accumulation. Ccll~ were incubated for 3 min in the presence of carbachoI (A} or histamine (B) at the indicated concentrations. For each concentration, the control value were deducted. Each point is the mean of two experiments. Note that A and B have different ordinates,
191 The curves of both agonists lay in the concentration range between 10 v and 10-4M; however, differences were obvious between the curves. Indeed, at a concentration of 10 7 M, carbachoI induced a marked accumulation of [3H]IPs, whereas the effect of histamine was undetectable. Both agonists were equipotent at an approximate concentration of 2 l0 -6 M. Histamine was conspicuously more effective than carbachol at higher concentrations: at 10 -4 M , the stimulation by histamine was four times larger than that of carbachol. The specificity of the action of these agonists was assessed by using specific antagonists; the response to carbachol, 10 5 M, was diminished by 67 + 2,5% (n = 4) in the presence
cpm/mg DNA (x I0 3)
A
of the muscarinergic antagonist atropine (1 raM) and the stimulatory effect of histamine (10 -5 M) was reduced by 90 _+ 6% (n = 4) by diphenhydramine (1 mM) an inhibitor of Hj-receptors. The [3H]IP accumulation stimulated by histamine, PGFza and carbachol was examined for selected [3H]IPs. Figure 6 shows the data observed for [3H]I-IP, [3H]4-IE [3H]I,4-IP2 and [3H)]IP3, following a 10-rain incubation period with or without agonist. The first [3H]IP3 peak to appear in the chromatogram was not altered in magnitude by either agent; similarly, the magnitude at the peaks corresponding to [3H]IP5 and [3H]IP6 varied insignificantly and these data were not included in figure 6. PGF2~ (10 -5 M) stimu-
cpm/mg DNA (x 10 3)
[3H]I-IP1
[31t]4-IP1
800
2800 T
2400
600
1200
ji
400 200
o
B
| (4)
(4)
(4)
(4)
80O 400 (3)
b
8
0
(3)
-
c
cpm/mg D N A (x I0 3)
C
(4) (4)
(4) (4)
(3) (3)
a
b
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cpm/mg D N A (x I0 3) [3H]IP3
[3H]I,4-IP2
400
D
80 T
300
60
200
40
100 0
20
i (4) (4) a
(4) (4) b
(3) (3) c
0
(4) (4) a
(4) (4) b
(3) (3) e
Fig 6. Production of selected lPs evoked by three agonists of contraction. A. I 3 H ] I - I P I . B . [3H]4-IPI.C. [3H]l,4-1P2.D. [3H]IP3. Cells were incubated for 10 min in the presence of 10-4 M histamine (a), 10-~ M carbachol (b) or 5 10-5 M PGFza (c). The hatched bars show the control values and the open bars show the values following agonist stimulation. Bars depict the mean + SEM of the number of observations indicated in brackets.
192 lated the production of total IPs from 1023 _+ 15 to 1655 + 58 thousands cpm/mg DNA, an effect of similar magnitude compared with that of carbachol. However, following 10 min of incubation, the amount of IP3 generated was almost totally degraded and the metabolites are depicted in figure 6. The major compounds to accumulate were IPi isomers because the recirculation in myo-inositol was prevented by Li. The accumulation of 1,4-IP2 and of IP3 isomers was not significantly affected by carbachol and PGFe~s, probably because the rate of degradation kept pace with that of formation. However, the accumulation of these compounds was revealed by the more potent agonist histamine. Taken together the present results with SMC from human airways in culture indicate that agonist stimulation elicits a breakdown of phosphoinositides that triggers the inositol phosphates cascade and thus the processing of the Ca-+-mo bilizing 1,4,5-IP3.
Discussion
Until recently, few details about the cellular or molecular mechanisms of action of bronchoconstrictor agents in human airway smooth muscle were available, because of the lack of a suitable preparation for these studies. The development of a cell culture system would certainly overcome the limitations inherent to muscle strips or freshly dispersed cells and would also provide the prominent advantage of yielding conspicuous amounts of material required for biochemical analysis. However, cells in culture can possibly undergo modulation leading to phenotypic modifications and loss of some specific physiological properties; thus a cell culture system may exhibit an altered functional state and the properties of the model must be unambiguously characterized in order to prove useful for further experirnentation. Morphological and functional properties of smooth muscle cells from animal airways or human bronchi in culture were described: Avner et al (1981) and Tom-Moy et al (1987) reported a method for culturing tracheal SMC of the dog. Cells in culture were found to contract following carbachol administration and to retain in subculture morphological characteristics of smooth muscle (Avner et al, 1981). More recently, Twort
and van Breemen (1988, 1989), Panettieri et al (1989) and Murray and Kotlikoff ( 1991 ) described a cell culture preparation from human airways that retained morphological and physiological responsiveness. Bronchial SMC obtained during lung resection were cultured and this preparation was found to respond to histamine and to carbachol by an elevated Ca 2+ release (Twort and van Breemen, 1988, 1989). In the present study, SMC from human bronchi in culture were found to exhibit growth characteristics and morphological properties similar to those described by previous authors for cultured SMC. In addition, results showed that these cells retained the ability to respond to histamine, carbachol and PGFe~ by an augmented production of lPs. This effect was dose-dependent and blocked by specific antagonists. In fact, it was the aim of this study to characterize in these cells the signal transduction system that is known Io trigger Ca 2+ release i'rom the sarcoplasmic reticulum. The inositol phospholipid transduction system involves as major components (Schramm and Grunstein, 1992): i) the cell surface receptor, ii) a G protein that is activated by the formation of the agonist-receptor complex and, in turn, activates (iii) the phospholipase C, which catalyses the breakdown of phosphatidylinositol 4,5-bisphosphate, thus producing (iv) two second messengers, namely diacylglycerol and 1,4,5-1P~. The latter mobilizes Ca "+ from the sarcoplasmic reticulum (Berridge and Irvine, 1989). It is well documented that an elevation in cytosolic Ca -'+ concentration is a key feature initiating smooth muscle contraction (Gerthoffer, 1991: Kamm and Siull, 1989: van Breemen and Saida, 1989: Coburn and Baron, 1990; De Lanerolle and Paul, 1991). The initiation of the contractile response to various agonists is currently thought Io resuh either from electromechanical coupling, a process linked to membrane depolarization, or from pharmacomechanical coupling, which is essentially independent from the membrane potential (Abdel-Latif, 1991). While the first inechanism prevails in vascular and intestinal smooth muscle, available evidence suggests that, in airway smooth muscle, pharmacomechanical coupling is the predominant mechanism linking the activation by an agonist of cell surface receptors to lhc mobilization of Ca -,+ from intracellular stores, thus eliciting the development of tension. Few data arc available on the possible role of w)ltage-dependent or receptor-operated Ca 2+ channels resident in the plasma membrane of airway SMC (Coburn
193 and Baron, 1990). In this respect, Marthan et a l (1989) s u g g e s t e d a role for v o l t a g e - d e p e n d e n t Ca 2+ channels. M u r r a y and K o t l i k o f f (1991) obs e r v e d that the time course o f the i n t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n during s t i m u l a t i o n by various a g o n i s t s f o l l o w e d a b i m o d a l pattern: a peak appeared in the first seconds, then the signal d e c r e a s e d and r e a c h e d a sustained level a b o v e the control value, lasting for several minutes; they p r o p o s e d that r e c e p t o r - a c t i v a t e d Ca 2+ channels were r e s p o n s i b l e for the sustained i n c r e a s e in the i n t r a c e l l u l a r Ca -,+ concentration. H o w e v e r , the p r o d u c t i o n o f IPs has not been r e p o r t e d so far in human b r o n c h i a l S M C . The present study was focused on the a b i l i t y of three a g o n i s t s o f b r o n c h o c o n s t r i c t i o n to stimulate IP m e t a b o l i s m , thus eliciting I P 3 - i n d u c e d Ca ~+ release in these cells. The results show that the s t i m u l a t i o n o f cell surface r e c e p t o r s for histamine, c a r b a c h o l and PGF2c~ resulted in an a u g m e n t e d p r o d u c t i o n o f IP s e c o n d m e s s e n g e r s in h u m a n a i r w a y S M C , suggesting that the c o u p l i n g b e t w e e n the receptors, the G proteins and the p h o s p h o l i p a s e C was functional. Twort and van B r e e m e n (1989) reported that the Ca 2÷ efflux s t i m u l a t e d by c a r b a chol was l o w e r than that i n d u c e d by the same c o n c e n t r a t i o n o f h i s t a m i n e and they s u g g e s t e d that the e x p r e s s i o n o f m u s c a r i n i c r e c e p t o r s was p a r t i a l l y lost in culture. Panettieri et a l (1989) observed a s i z e a b l e Ca 2+ release f o l l o w i n g histamine a d m i n i s t r a t i o n , but the r e s p o n s e to m e t h a c h o l i n e was absent. F r e s h l y isolated cells were shown to contract in r e s p o n s e to both h i s t a m i n e and a c e t y l choline (Marthan et al, 1989). During the present study, we o b s e r v e d that c a r b a c h o l at r e l a t i v e l y high c o n c e n t r a t i o n s was less effective than h i s t a m i n e in e n h a n c i n g the IP production. However, it is interesting to note that the d o s e - r e sponse curves e s t a b l i s h e d during the present study r e v e a l e d that c a r b a c h o l in the low c o n c e n t r a t i o n range was indeed m o r e potent than h i s t a m i n e to elicit the a c c u m u l a t i o n of IPs, s u g g e s t i n g that the m u s c a r i n i c r e c e p t o r s r e t a i n e d a high affinity for the agonist. In c o n c l u s i o n , human a i r w a y S M C in culture were used as an in vitro m o d e l to investigate the p o s t - r e c e p t o r events i n v o l v e d in agonist stimulation; the p h y s i o l o g i c a l s i g n i f i c a n c e o f this approach is s u g g e s t e d by the o b s e r v a t i o n that d i v e r s e b r o n c h o a c t i v e m e d i a t o r s caused an increase in PLC activity, thus o p e n i n g the possibility to investigate in human a i r w a y S M C the m e c h a n i s m s o f m e m b r a n e t r a n s d u c t i o n and o f the p h y s i o l o g i c a l control o f contraction.
Acknowledgments We wish to thank Drs J Savoy, P Hahnloser and P Petropoulos from the H6pital cantonal de Fribourg, for the obtainment of bronchi segments. The excellent technical help of J Ruffieux is gratefully acknowledged. We thank P Haab of our Institute for stimulating discussions. The work was supported by the Swiss National Foundation, grant 32-27825.89.
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