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Effect of vascular smooth muscle relaxants on the protein kinase C-mediated contraction in the rat pulmonary artery
European Journal of Pharmacology, 249 (1993) 191-198
191
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53373
Effect of vascular smooth muscle relaxants on the protein kinase C-mediated contraction in the rat pulmonary artery J e a n - P i e r r e S a v i n e a u *, P a t r i c k G o n z a l e z D e L a F u e n t e a n d R o g e r M a r t h a n Laboratoire de Physiologie, Universit~ de Bordeaux II, 146 Rue L~o Saignat, 33076 Bordeaux Cedex, France
Received 19 April 1993, revised MS received 29 July 1993, accepted 24 August 1993
In the rat pulmonary artery, phorbol 12,13-dibutyrate induces a contraction due to the activation of the protein kinase C. We investigated the sensitivity of this protein kinase C-mediated contraction to a variety of vascular smooth muscle relaxants. Pretreatment of rat pulmonary artery with relaxant compounds altered the subsequent concentration-response curve to phorbol 12,13-dibutyrate (0.05-2/xM) in a variable manner. Isoprenaline (0.1-10/zM), nifedipine (0.01-1 /zM) and cromakalim (0.1-10 /zM) had no effect, whereas vasoactive intestinal peptide (VIP, 1-10 nM), forskolin (0.1-2 /xM), theophylline (0.1-2.5 mM), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20-1724, 2-20 /zM), dipyridamole (10-100 /xM), 8 bromo-cyclic GMP (8-br-cGMP, 5-500/zM) and dibutyryl cyclic AMP (db-cAMP, 100-500/~M) shifted the concentration-response curve to phorbol 12,13-dibutyrate to the right and decreased the maximal response. When cumulative concentrations of relaxants were applied on the plateau of the contraction induced by 0.2 or 2/xM phorbol 12,13-dibutyrate, again, isoprenaline, nifedipine and cromakalim failed to decrease the protein kinase C-mediated contraction, whereas the other agents produced concentration-dependent relaxation. From their inhibitory effect on the 0.2/zM phorbol 12,13-dibutyrate-induced contraction, the rank order of potency of these relaxants was: VIP >> forskolin > RO 20-1724 > 8-br-cGMP > theophylline > dipyridamole > db-cAMP. In chemically (fl escin) skinned preparations, cGMP (5-500/zM) and cAMP (50-1000/xM) antagonized in a concentration dependent manner the contraction induced by phorbol 12,13-dibutyrate at constant Ca z÷ concentration. These results show that protein kinase C-mediated contraction in pulmonary vascular smooth muscle is not altered by ion channel modulators or by stimulation of the fl-adrenoceptor with isoprenaline. On the other hand, protein kinase C-mediated contraction is reduced by other compounds which increase the intracellular cyclic nucleotide content. The interaction between protein kinase C and cyclic nucleotide-dependent protein kinases may be important in the physiological and pathophysiological control of tone in the pulmonary circulation. Pulmonary artery; Smooth muscle; Vascular relaxant; Phorbol ester; Protein kinase C; Phosphodiesterase inhibitor; Cyclic nucleotides
I. Introduction Activation of protein kinase C, an ubiquitous cellular enzyme (Nishizuka, 1986), may play a crucial role in the maintenance of tension in vascular smooth muscles stimulated by agonists (Rasmussen et al., 1987; Jiang and Morgan, 1987; Khalil and Morgan, 1992). This hypothesis is currently investigated using the tumour promoting phorbol esters which, easily, penetrate smooth muscles cells, bind to and activate protein kinase C (Kikkawa et al., 1983; Leach and Blumberg, 1989). Phorbol esters induce slow developing and sustained contraction in smooth muscles isolated from a variety of vascular beds (e.g. aorta, Jiang and Morgan, 1987; coronary arteries, Mori et aL, 1990; cerebral
* Corresponding author. Tel. 57 57 13 60, fax 56 99 03 80.
arteries, Salaices et al., 1990; saphenous vein, Chiu et al., 1988). Such is also the case in the pulmonary artery (Orton et al., 1990; Savineau et al., 1991). In the pulmonary circulation, protein kinase C has also been implicated in pathophysiological processes such as pulmonary hypertension (Marche, 1989; Dempsey et al., 1991) and hypoxic vasoconstriction (Orton et al., 1990; Jin et al., 1992). If, at least a part of these processes is linked to an abnormal activation of protein kinase C, it would be relevant to investigate the effect of smooth muscle relaxants on the protein kinase C-mediated contraction in the pulmonary circulation. The interaction between some relaxants and protein kinase C has been previously studied in the guinea-pig lung parenchymal strip (Obianime et al., 1989) which contains distal airway, microvascular and other contractile cells. However, direct investigation of this interaction in the isolated pulmonary artery has not yet been performed.
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We therefore designed the present study to investigate the effect of ten vascular relaxants on the contractile response elicited by phorbol 12,13-dibutyrate, a potent protein kinase C-activator (Castagna et al., 1982) in the main pulmonary artery of the rat (Savineau et al., 1991). Relaxants were chosen to act via various pathways in the relaxation process. We thus have studied responses to ion channel modulators (nifedipine, cromakalim), membrane receptor agonists (isoprenaline, vasoactive intestinal peptide (VIP), an adenylate cyclase activator (forskolin), phosphodiesterase inhibitors (theophylline, 4-(3-butoxy-4-methoxybenzyl)-2imidazolidinone (RO 20-1724), dipyrimadole) and cyclic nucleotide analogues (8-bromo-cyclic GMP (8-brcGMP), dibutyryl cyclic AMP (db-cAMP)). We also loocked at the effect of cyclic AMP (cAMP) and cyclicGMP (cGMP) on the phorbol 12,13-dibutyrate-induced contraction in chemically (/3 escin) skinned preparations.
2. Materials and methods
2.1. Tissue preparation Experiments were performed on strips (200-400 tzm in diameter, 8-10 mm in length) isolated from the main pulmonary artery of male Wistar rats aged from 10 to 15 weeks. Animals were anesthetized by intraperitoneal injection of 40 mg sodium pentobarbitone. Heart and lungs were removed en-bloc. The main pulmonary artery was then rapidly dissected and opened. Strips (two to three for each artery) were cut transversely.
2.2. Mechanical recording Isometric contraction was measured in an experimental chamber (Savineau et al., 1988) by means of a highly sensitive force transducer (Akers 801, Horten, Norway) connected to a micromanipulator allowing to stretch the strip to a length close to the optimal length determined in preliminary experiments.
2.3. Solutions 2.3.1. Solutions for intact tissues Physiological solutions had the following composition. Reference solution (mM): NaCl 118.4, KCl 4.7, CaC12 2.5, MgSO4 1.2, KHEPO 4 1.2, NaHCO 3 25, glucose 11; the solution was aerated with 5% CO 2 in 0 2. A K+-rich (80 mM) solution was prepared by substituting an equimolar amount of KC1 for NaC1.
2.3.2. Solutions for skinned tissues The main relaxing solution used in these experiments had the following composition (mM): KCI 85, MgCI 2 5, ATP (Na) 2 5, EGTA 10, Tris 20, maleic acid 20 brought to pH 7.1 at 25°C with KOH. Solutions with variable free Ca 2÷ concentration were also prepared by mixing appropriate amounts of standard Ca 2÷ and EGTA solutions. The free Ca 2÷ concentration was calculated using a computer programm adapted from that of Fabiato (1988). The ionic strength of the solution was standardized at 0.2 M by adjusting the KC1 concentration. 2.4. Experiments with intact tissues 2.4.1. Pretreatment of the tissues with relaxants on the phorbol 12,13-dibutyrate-induced response At the beginning of the experiments, K+-rich (80 mM) solution was repeatedly applied in order to obtain at least three contractions similar in both amplitude and kinetics before the addition of phorbol 12,13-dibutyrate. A control concentration-response curve to phorbol 12,13-dibutyrate was constructed by adding cumulative concentrations of phorbol 12,13-dibutyrate (0.05-2 tzM). A concentration increment was made once the maximal contractile effect of the preceding concentration had been recorded (generally 10-12 min). A second then a third cumulative concentrationresponse curve to phorbol 12,13-dibutyrate was constructed in the presence of two different concentrations of one relaxant, applied for 15 min previously. It was verified, in control experiments, that three or even four identical concentration-response curves to phorbol 12,13-dibutyrate could be obtained from the same arterial strip provided that a time interval of 1 h was allowed between two successive cumulative concentration-response curves. The response to each concentration of phorbol 12,13-dibutyrate alone or of phorbol 12,13-dibutyrate in the presence of the relaxant was expressed as a percentage of the maximal response obtained in the first cumulative concentration-response curve.
2.4.2. Effect of relaxants on tissues precontracted with phorbol 12,13-dibutyrate In a second set of experiments, pulmonary artery strips were precontracted with 0.2 and 2 ~M phorbol 12,13-dibutyrate, concentrations inducing half-maximal and maximal responses, respectively as determined from the above experiments. When the contractile response plateaued, concentration-response curves to relaxants were constructed by adding cumulative concentrations of the compounds. A concentration increment was made once the maximal relaxant effect of the preceding concentration had been recorded: usually 8-10 min with forskolin or theophylline, and 15-18
193 min with isoprenaline, VIP, RO 20-1724, dipyridamole, db-cAMP, 8-br-cGMP, nifedipine or cromakalim. It was verified, in control experiments, that the contraction induced by phorbol 12,13-dibutyrate alone was maintained as long as 1 h without significant variation in amplitude (1.7 + 0.2% decrease in the amplitude of 0.2 txM phorbol 12,13-dibutyrate-induced contraction between 15 and 60 min, n --- 7, P > 0.05). The response to each concentration of relaxant was expressed as a percentage of the contractile response elicited by 0.2 or 2 /zM phorbol 12,13-dibutyrate alone. A mean curve was constructed which enabled the concentration producing a decrease of 50% of the phorbol 12,13-dibutyrate-induced contraction to be determined. Since it has been previously shown that phorbol 12,13-dibutyrate-induced contraction is endothelium independent (Savineau et al., 1991), experiments with these tissues were performed on endothelium intact pulmonary artery strips at 37 + 0.5°C.
were more consistent in amplitude when temperature was decreased below 30°C.
2.5. Experiments with /3 escin-treated tissues
2. 7. Analysis of results
Arterial strips used in these experiments were dissected as mentioned above and in addition were cleaned of their periarterial connective tissue and scratched on the luminal surface to remove the endothelium. After recording the contractile response to the K+-rich (80 raM) solution, the strip was incubated in the main relaxing solution in the presence of 80/~M /3 escin for 30 rain. During the 15 latter min of this skinning period, the ionophore A23187 (10 /xM) was also added to impair the Ca 2÷ accumulating and releasing functions of the sarcoplasmic reticulum. After removal of/3 escin and A23187 the success of skinning was attested by recording a contraction to 20/xM free Ca 2+ ions the amplitude of which was higher (115 + 10.4%) than that induced by K ÷ rich (80 mM) solution before the skinning procedure. Then, after complete relaxation, the Ca 2÷ concentration was adjusted to 0.08 /xM (pCa 7.1). Phorbol 12,13-dibutyrate (0.2 or 2 ~M) was applied inducing a maintained contraction. At the plateau of the response, a concentration-response curve to cAMP or cGMP was performed by adding cumulative concentration of the compounds. One concentration response curve was established for each /3 escintreated strip. The effect of the ionophore A23187 on the functioning of the sarcoplasmic reticulum was checked by comparing the contractile effect of inositol 1,4,5-trisphosphate in tissues exposed or unexposed to A23187. After the skinning procedure, the EGTA concentration was lowered to 0.1 mM and 0.5 /xM free Ca 2+ were applied for 8 min to load the sarcoplasmic reticulum. 10/zM inositol 1,4,5-trisphosphate was then added to the solution. Experiments with/3 escin-treated tissues were done at 25°C, since we observed, in preliminary experiments, that CaZ+-induced contractions
The amplitude of contractions is expressed as mean + S.D. for n, number of experiments. Significance was assessed by paired Student's t-tests. A difference between means was considered significant when P < 0.05.
2.6. Chemicals Chemicals used in the present study were: cAMP, db-cAMP, cGMP, 8-br-cGMP, dipyridamole, forskolin, inositol 1,4,5-trisphophate, isoprenaline, phorbol 12,13-dibutyrate, theophylline, VIP all from Sigma (La Verpill~re, France); RO 20-1724 and nifedipine from Gibco BRL (Cergy Pontoise, France); cromakalim was a gift from Smith Kline Beecham Research pharmaceuticals (Betchworth, U.K.). Dipyridamole, forskolin, nifedipine and phorbol 12,13-dibutyrate were dissolved in dimethylsulfoxide (DMSO), and cromakalim was dissolved in 70% ethanol. The maximal concentration of DMSO or ethanol in the bath was < 0.1% and had no effect on the contractility of main pulmonary artery strips.
3. Results
3.1. Effect of pretreatment of tissues with relaxants on the cumulative concentration-response curve to phorbol 12,13-dibutyrate Fig. 1 shows examples of the effect, on the pulmonary arterial tone, of cumulatively applied concentrations of phorbol 12,13-dibutyrate (0.05-2 ~M) alone (ABC, a) and of phorbol 12,13-dibutyrate in the presence of isoprenaline (10 /~M, Ab), theophylline (500 tzM, Bb) and db-cAMP (500 /xM, Cb). The results obtained with the ten relaxants tested are shown on figs. 2-4. VIP (1-10 nM), forskolin (0.1-2 /zM), RO 20-1724 (2-20 /zM), dipyridamole (10-100 /~M) theophylline (0.1-2.5 mM), 8-br-cGMP (5-500 /zM) and db-cAMP (100-500/zM) all induced a rightward shift of the cumulative concentration-response curve with a decrease in the maximal response (figs. 2, 3). For example, 2 IzM forskolin or 500/~M theophylline - the two most efficacious compounds - reduced 2 /~M phorbol 12,13-dibutyrate-induced contraction by 88.2 + 3.7% (n = 5) and 80.6 + 6.1% (n = 5), respectively, whereas 10 nM VIP reduced this contraction by only 36 + 7.8% (n = 5). Higher of concentrations VIP (50 and 100 nM) had 'no further effect on the cumulative concentration-response curve to phorbol 12,13-dibutyrate (n = 6, not shown). On the contrary, the cu-
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Fig. 1. Original traces showing the effect of three vascular relaxants on contractions induced by phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentrations (0.05-2 ~ M ) of phorbol 12,13-dibutyrate (PDB) were applied in control tissues (ABCa) and in tissues pretreated with isoprenaline (10 p.M, Ab), theophylline (500/~M, Bd) and db-cAMP (500 /.LM, Cb). Relaxants were applied 15 min before and throughout the concentration-response curve for phorbol 12,13-dibutyrate.
mulative concentration-response curve to phorbol 12,13-dibutyrate was nearly unaltered in the presence of isoprenaline (0.1-10/xM, fig. 2A), nifedipine (0.01-1 /zM, fig. 4A) or cromakalim (0.1-10/zM, fig. 4B).
3.2. Effect of relaxants on tissues precontracted with phorbol 12,13-dibutyrate Main pulmonary artery strips stimulated with 0.2 /zM (fig. 5A) or 2 /zM phorbol 12,13-dibutyrate developed a contractile response which reached a plateau in 15 + 3.2 min ( n = 12) and 18.5 + 2.8 min ( n = 10), respectively, and could be maintained without significant change in amplitude for as long as 1 h. Removal of TABLE 1 Concentrations (~.M) of relaxants producing 50% decrease in the 0.2 and 2 p.M phorbol 12,13-dibutyrate-induced contractions. ( - ) = not determined. Values were determined from a m e a n curve obtained from five experiments for each relaxant. Relaxant
Isoprenaline VIP Forskolin RO-201724 Dipyridamole Theophylline 8-br-cGMP db-cAMP Nifedipine Cromakalim
Phorbol 12,13-dibutyrate 0.2/zM
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Fig. 2. Effect of cyclic nucleotide-elevating c o m p o u n d s on the cumulative concentration-response curve for phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentration-response curve was obtained from control tissues (e) and from tissues pretreated with: (A) isoprenaline (10/zM, • ) ; (B) VIP (1 nM * , 10 nM II); (C) forskolin (0.1 /zM • , 1 / z M II, 2 / ~ M o); (D) theophylline (100/~M • , 250/xM III, 5 0 0 / z M o); (E) R O 20-1724 ( 2 / x M • , 10 /zM II, 20 /xM o ) and (F) dipyridamole (10 • , 50 II, 100/~M o). In each panel, abscissa scale: - log concentration (M) of phorbol 12,13-dibutyrate (PDB) and ordinate scale: tension as a percentage of the maximal response induced by 2 /zM phorbol 12,13-dibutyrate in control tissues. Data points are mean, n = 5. Vertical lines show S.D. * indicates a response in the presence of relaxant which is significantly different ( P < 0.05) from the control experiment.
phorbol 12,13-dibutyrate induced a slow recovery of the basal tone which re-established in 25 + 3.2 min (n = 12) and 41 + 4.5 min (n --- 10) for 0.2 and 2 /zM phorbol 12,13-dibutyrate, respectively. Fig. 5BC shows an example of the effect of cumulatively applied concentrations of 8-br-cGMP and of 10 /zM cromakalim, respectively on the 0.2 /zM phorbol 12,13-dibutyrateinduced contraction. Concentrations of relaxants producing a 50% decrease in the contraction induced by 0.2 and 2 /xM phorbol 12,13-dibutyrate are listed in table 1. From their respective effect on tissues precontrated with 0.2 /zM phorbol 12,13-dibutyrate, the rank
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Fig. 3. Effect of cyclic nucleotide analogues on the cumulative concentration-response curve for phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentration-response curve was obtained from control tissues (e) and from tissues pretreated with: (A) db-cAMP (100 tzM • , 250 p.M I , 500 /zM ©) and (B) 8-br-cGMP (10 p.M • , 100 tzM I ) . In each panel, abscissa scale: - l o g concentration (M) of phorbol 12,13-dibutyrate (PDB) and ordinate scale: tension as a percentage of the maximal response induced by 2 /zM phorbol 12,13-dibutyrate in control tissues. Data points are mean, n = 5. Vertical lines show S.D. * indicates a response in the presence of analogue which is significantly different from the corresponding response in the control experiment.
Fig. 5. Effect of vascular relaxants on the rat isolated pulmonary artery pre-contracted with phorbol 12,13-dibutyrate. (A) Control contraction induced by 0.2/xM phorbol 12,13-dibutyrate (PDB). (B) Effect of cumulatively applied concentrations of 8-br-cGMP. (C) Effect of 10/zM cromakalim applied for the time indicated by the dotted line.
order of potency of relaxants appeared to be: VIP >> forskolin > RO 20-1724 > 8-br-cGMP > theophylline > dipyridamole > db-cAMP. Again in these experiments, isoprenaline (0.1-10 /~M, n =5), nifedipine (0.1-5 /zM, n = 4) and cromakalim (1-10 IzM, n = 4) failed to relax phorbol 12,13-dibutyrate-precontracted pulmonary artery. However, in similar experiments, 1 /.~M isoprenaline produced 50.3 + 4.2% (n = 5) relaxation in 80 mM KCl-precontracted tissues and propranolol (5 ~M) abolished this effect (data not shown).
tractile response, application of 0.2 (fig. 6Ba) or 2/xM phorbol 12,13-dibutyrate induced a sustained contraction the amplitude of which was 54.3 + 6.8 and 122 + 14.2% (n = 4) respectively of that produced by 20/xM Ca 2÷. The amplitude and kinetics of the phorbol 12,13-dibutyrate-induced contraction was not modified in arterial strips unexposed to the ionophore A23187 (fig. 6Aa). On the other hand, the contractile response to inositol 1,4,5-trisphosphate (10 ~M) was dependent on the pretreatment of the strip with the ionophore
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3.3. Experiments in [3 escin-treated tissues When the Ca 2+ concentration was clamped to 0.08 /zM a concentration which by itself produced no con-
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Fig. 4. Effect of ion channel modulators on the cumulative concentration-response curve for phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentration-response curve was obtained from control tissues (e) and from tissues pretreated with: (A) nifedipine (1 /zM, • ) and (B) cromakalim (10 g,M, • ) . In each panel, abscissa scale: - l o g concentration (M) of phorbol 12,13-dibutyrate (PDB) and ordinate scale: tension as a percentage of the maximal response induced by 2 /zM phorbol 12,13-dibutyrate in control tissues. Data points are mean, n = 5. Vertical lines show S.D.
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Fig. 6. Effect of pretreatment with ionophore A23187 on the contraction induced by phorbol 12,13-dibutyrate and inositol 1,4,5-trisphosphate in fl escin skinned tissues. In tissues unexposed to A23187 (top panel A), phorbol 12,13-dibutyrate (PDB) and inositol 1,4,5-trisphosphate (IP 3) induce a contraction (Aa and Ab, respectively). In tissues exposed to A23187 (10/zM) applied during the last 15 min of the skinning procedure (lower panel B), contraction to phorbol 12,13-dibutyrate is unchanged (Ba) whereas contraction to inositol 1,4,5-trisphospbate is abolished (Bb). Prior stimulation with inositol 1,4,5-trisphosphate, the sarcoplasmic reticulum was loaded with 0.5 /zM Ca 2÷ for 8 rain. This result is typical of four experiments.
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Fig. 7. Effect of cyclic nucleotides on the phorbol 12,13-dibutyrateinduced contraction in /3 escin-treated preparations. Concentrationresponse curves for the effect of cGMP (A) and cAMP (B) on contractions induced by 0.2 (e) and 2 p.M ( a ) phorbol 12,13-dibutyrate, respectively. For each panel, abscissa scale shows - l o g concentration (M) of cyclic nucleotide and ordinate scale gives tension as a percentage of the phorbol 12,13-dibutyrate-induced contraction in the absence of cyclic nucleotide. Data points are mean, n = 4. Vertical lines show S.D. The free Ca 2÷ concentration was kept constant at 0.08/zM thoughout the experiments.
A23187 (fig. 6ABb). Further addition of cGMP (1-500 /xM) or cAMP (10-1000 /zM) at the plateau phase of the phorbol 12,13-dibutyrate response induced a concentration-dependent inhibition of this contraction (fig. 7). The concentrations producing 50% decrease of the 0.2 and 2 jzM phorbol 12,13-dibutyrate-induced contraction were 12 and 30 ~M cGMP; 120 and 210 ~M cAMP, respectively.
4. Discussion
Our study shows that the alteration in phorbol 12,13-dibutyrate-induced contraction in rat main pulmonary artery depends on the type of smooth muscle relaxants used. The phorbol 12,13-dibutyrate-induced contraction is antagonized by adenylate cyclase activator (forskolin), cyclic nucleotide phosphodiesterase inhibitors (theophylline, RO 20-1724, dipyridamole) and cyclic nucleotide analogues (db-cAMP, 8-br-cGMP), whereas it is not modified by ion channel modulators (nifedipine, cromakalim). Finally, relaxants acting at membrane receptors (isoprenaline, VIP) exhibit peculiar effect according to the type of the stimulated receptor. Previous studies (Orton et al., 1990; Savineau et al., 1991) have shown that phorbol 12,13-dibutyrate-induced contraction in the rat pulmonary artery is due to the activation of protein kinase C and is independent of both Ca 2÷ influx and Ca 2÷ release from internal stores. The fact that the Ca 2+ channel antagonist nifedipine and the potassium channel opener cromakalim (fig. 4) did not alter phorbol 12,13-dibutyrateinduced contraction whereas they inhibited KCl-induced contraction in this same preparation (Orton et al., 1990; Savineau and Marthan, 1993) corroborates
the fact that protein kinase C-mediated contraction is a membrane potential-independent process. Moreover, the fact that phorbol 12,13-dibutyrate-induced contraction in skinned tissues is similar whether the sarcoplasmic reticulum is functional or not (fig. 6) confirms that it is independent of Ca 2÷ release from internal stores. These findings suggest that the phorbol 12,13-dibutyrate-stimulated enzyme is a Ca-independent isoform of protein kinase C as also shown in the rat aorta (Collins et al., 1992). The main result of the present study is that protein kinase C-mediated contraction in the pulmonary circulation is sensitive to substances which increase or mimick intracellular cyclic nucleotides at a site distal to the membrane receptor. Among these relaxants, forskolin an activator of the catalytic subunit of adenylate cyclase (Seamon and Daly, 1983) appears the most potent (fig. 2C and table 1). This result is similar to those of Obianime and Dale (1989) and of Salaices et al. (1990), in rat aorta and cat cerebral arteries, respectively. Theophylline, a non-specific phosphodiesterase inhibitor, increases both the cAMP and cGMP content of smooth muscle cells (Weishaar et al., 1986) while RO 20-1724 and dipyridamole are specific inhibitors of cAMP-dependent and cGMP-dependent phosphodiesterase, respectively (Schoeffter et al., 1987; Ahn et al., 1992). Since the detailed mechanism by which protein kinase C contracts vascular smooth muscle is not yet fully elucidated, it is difficult to explain the mode of action of cyclic nucleotides on this protein kinase Cmediated contraction. Protein kinase C has been shown to phosphorylate a variety of proteins such as myosin light chains (Nishikawa et al., 1983; Singer, 1990), caldesmon (Adam et al., 1989), calponin (Winder and Walsh, 1990) and the myristorylated alanine rich C kinase substrate (Hartwig et al., 1992) which are involved in the control of the smooth muscle contractile apparatus. How cyclic nucleotides interact with these proteins is a matter of debate. However, the effect of cAMP and cGMP on the phorbol 12,13-dibutyrate-induced contraction in /3 escin-treated tissues (fig. 7) suggests that cAMP and cGMP-dependent protein kinases decrease whereas protein kinase C increases the sensitivity of the contractile machinery in pulmonary artery as previously shown in other vascular beds (Jiang and Morgan, 1987; Itoh et al., 1985; Nishimura and Van Breemen, 1989). Thus a direct effect of these kinases on the contractile apparatus could be the molecular basis for the interaction and the regulatory action of protein kinases on vascular smooth muscle tone. A paradoxical result of our study is the absence of effect of isoprenaline, a specific /3-adrenoceptor agonist, on phorbol 12,13-dibutyrate-induced contraction. This lack of effect cannot be ascribed to the absence or the non-functioning of/3-adrenoceptors in the rat main
197
pulmonary artery since it has been shown that the /3-adrenoceptor density in pulmonary artery is higher than in aorta (Shaul et al., 1990) and similar to that in the bronchial smooth muscle (Schell et al., 1992). Moreover isoprenaline relaxes KCl-precontracted pulmonary artery (O'Donell and Wanstall, 1984; present study). On the other hand, the coupling between the increase in cyclic AMP content and fl-adrenoceptor ligand binding (Shaul et al., 1990) is reduced by phorbol 12,13-dibutyrate which decreases by approximately 50% the cyclic AMP production in response to isoprenaline without altering its Bmax (Aiyar et al., 1987). Phorbol ester-activated protein kinase C phosphorylates /3-adrenoceptors (Sibley et al., 1984), thus inducing an heterologous desensitization without internalization of these receptors (Aiyar et al., 1987; Toews et al., 1988). The resulted uncoupling between /3-adrenoceptors and adenylate cyclase could be responsible for the insensitivity to isoprenaline of the protein kinase Cmediated contraction in the rat main pulmonary artery as also observed in the rat aorta (Obianime and Dale, 1989). The other receptor agonist used in the present study was VIP, a transmitter of the non-adrenergic noncholinergic system in the pulmonary tract (Barnes, 1988). VIP receptors have been localized on the smooth muscle membrane of pulmonary vessels (Dey et al., 1981; Laitinen et al., 1985), and VIP is a potent relaxant of isolated pulmonary arteries precontracted with prostaglandin H 2 (Hamasaki et al., 1983). The potency of VIP on the protein kinase C-mediated contraction was high (in the nM range) (table 1) but its efficacy (fig. 2B) was limited even for the highest tested concentration (100 riM). This limited efficacy could be explained by the fact that the mechanism underlying VIP action is only partly related to an increase in cAMP content (Schoeffter and Stoclet, 1985) or/and by a desensitization of the receptor similar to that of /3-adrenoceptors. In conclusion, our study has shown that protein kinase C-mediated vasoconstriction in the pulmonary circulation can be reversed by substances which by-pass the receptor agonist step and bring about an increase in the cyclic nucleotide content of vascular smooth muscle cells. Thus, interactions between protein kinase C and cyclic nucleotide-dependent protein kinases may play a role in the control of pulmonary vascular tone. Whether these interactions would be altered in pathological processes involving the pulmonary circulation merits further investigations. Acknowledgements The authors thank Ms H. Crevel for technical assistance and Ms M.C. Chauvet for typing the manuscript. This work was supported by a grant from 'Conseil R6gional d'Aquitaine' (no. 9203023).
References Adam, L.P., J.R. Haeberle and D.R. Hathaway, 1989, Phosphorylation of caldesmon in arterial smooth muscle, J. Biol. Chem. 264, 7698. Ahn, H.S., W. Crim, B. Pins and E.J. Syhertz, 1992, Calciumcalmodulin-stimulated and cyclic-GMP-specific phosphodiesterases, in: Advances in Second Messenger and Phosphoprotein Research, eds. S.J. Strada and H. Hidaka (Raven Press, New York) p. 271. Aiyar, N., P. Nambi, M. Whitman, F.L. Stassen and S.T. Crooke, 1987, Phorbol ester-mediated inhibition of vasopressin and fladrenergic responses in a vascular smooth muscle cell line, Mol. Pharmacol. 31, 180. Barnes, P.J., 1988, Neuropeptides in human airways, in: Mechanisms in Asthma: Pharmacology, Physiology and Management, eds. C.L. Armour and J.L. Black, Alan R. Liss, New York) p. 111. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y. Nishizuka, 1982, Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor promoting phorbol esters, J. Biol. Chem. 257, 7847. Chiu, J.P.S., G. Tetzloff, M. Chatterjee and E.J. Sybertz, 1988, Phorbol 12,13 dibutyrate, an activator of protein kinase C, stimulates both contraction and Ca 2+ fluxes in dog saphenous vein, Naunyn-Schmied. Arch. Pharmacol. 338, 114. Collins, E.M., M.P. Walsh and K.G. Morgan, 1992, Contraction of single vascular smooth muscle cells by phenylephrine at constant ( Caa* )i, Am. J. Physiol. 262, H754. Dempsey, E.C., I.F. Mc Murtry and R.F. O'Brien, 1991, Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia, Am. J. Physiol. 260, L136. Dey, R.D., W.A. Shannon and S.I. Said, 1981, Localization of VIP immunoreactive nerves in airways and pulmonary vessels of dogs, cats and human subjects, Cell Tissue Res. 220, 231. Fabiato, A., 1988, Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands, in: Methods in Enzymology, Vol. 157, eds. S. Fleischer and B. Fleischer (Academic Press, Orlando) p. 378. Hamasaki, Y., H. Mojarad and S.I. Said, 1983, Relaxant action of VIP on cat pulmonary artery: comparison with acetylcboline, isoproterenol, and PGE1, J. Appl. Physiol. 54, 1607. Hartwig, J.H., M. Thelen, A. Rosen, P.A. Jaumey, A.C. Nairn and A. Aderem, 1992, MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin, Nature 356, 618. Itoh, T., Y. Kanmura, H. Kuriyama and T. Sasaguri, 1985, Nitroglycerine- and isoprenaline-induced vasodilatation: assessment from the actions of cyclic nucleotides, Br. J. Pharmacol. 84, 393. Jiang, M.J. and K.G. Morgan, 1987, Intracellular calcium levels in phorbol ester-induced contractions of vascular muscle, Am. J. Physiol. 253, H1365. Jin, N., C.S. Packer and R.A. Rhoades, 1992, Pulmonary arterial hypoxic contraction: signal transduction, Am. J. Physiol. 263, L73. Khalil, R.A. and K.U. Morgan, 1992, Protein kinase C: a second E-C coupling pathway in vascular smooth muscle, News Physiol. Sci. 7, 10. Kikkawa, U., Y. Takai, Y. Tanaka, R. Miyaker and Y. Nishizuka, 1983, Protein kinase C as a possible receptor protein of tumorpromoting phorbol esters, J. Biol. Chem. 258, 11442. Laitinen, A., M. Partanen, A. Hervonen, M. Peto-Huikko and L.A. Laitinen, 1985, VIP-like immunoreactive nerves in human respiratory tract. Light and electron microscopic study, Histochemistry 82, 313. Leach, K. and P.M. Blumberg, 1989, Tumour promotors, their receptors and their actions, in" Inositol Lipids in Cell Signalling, ed. R.M. Michell (Academic Press, New York) p. 179.
198 Marche, P., 1989, Membrane phosphoinositide metabolism in hypertension, News Physiol. Sci. 4, 230. Mori, T., T. Yanagisawa and N. Taira, 1990, Phorbol 12,13 dibutyrate increases vascular tone but has a dual action on intracellular calcium levels in porcine coronary arteries, Naumyn-Schmied. Arch. Pharmacol. 341, 251. Nishikawa, M., M. Hidaka and R.S. Adelstein, 1983, Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospolipid-dependent protein kinase, J. Biol. Chem. 258, 14069. Nishimura, J.and C. Van Breemen, 1989, Direct regulation of smooth muscle contractile elements by second messengers, Biochem. Biophys. Res. Commun, 163, 929. Nishizuka, Y., 1986, Studies and perspectives of protein kinase C, Science 233, 305. Obianime, A.W. and M.M. Dale, 1989, The effect of relaxants working through different transduction mechanisms on the tonic contraction produced in rat aorta by 4/3-phorbol dibutyrate, Br. J. Pharmacol. 97, 647. Obianime, A.W., S.J. Hirst and M.M. Dale, 1989, The effect of smooth muscle relaxants working through different transduction mechanisms on the phorbol dibutyrate-induced contraction of the guinea-pig lung parenchyma strip: possible relevance for asthma, Pulm. Pharmacol. 2, 191. O'Donnell, S.R. and J.C. Wanstall, 1984,/3-1 and/3-2 adrenoceptormediated responses in preparations of pulmonary artery and aorta from young and aged rats, J. Pharmacol. Exp. Ther. 228, 733. Orton, E.C., B. Raffestin and |.F. Mc Murtry, 1990, Protein kinase C infuences rat pulmonary vascular reactivity, Am. Rev. Respir. Dis. 141, 654. Rasmussen, H., Y. Takuwa and S. Park, 1987, Protein kinase C in the regulation of smooth muscle contraction, FASEB J. 1, 177. Salaices, M., G. Balfagon, S. Arribas, M.R. De Sagarra and J. Martin, 1990, Effects of phorbol 12,13 dibutyrate on the vascular tone and on norepinephrine- and potassium-induced contractions of cat cerebral arteries, J. Pharmacol. Exp. Ther. 255, 66. Savineau, J.P. and R. Marthan, 1993, Effect of cromakalim on KCI-, noradrenaline and angiontensin II-induced contractions in the rat pulmonary artery, Pulm. Pharmacol. 6, 41. Savineau, J.P., J. Mironneau and C. Mironneau, 1988, Contractile
properties of chemically skinned fibers from pregnant rat myometrium: existence of an internal Ca-store, Pfliigers Arch. 411, 296. Savineau, J.P., R. Marthan and H. Crevel, 1991, Contraction of vascular smooth muscle induced by phorbol 12,13 dibutyrate in human and rat pulmonary arteries, Br. J. Pharmacol. 104, 639. Schell, D.N., D. Durham, 5.5. Murphree, K.H. Muntz and P.W. Shaul, 1992, Ontogeny of fl-adrenergic receptors in pulmonary arterial smooth muscle, bronchial smooth muscle and alveolar lining cells in the rat, Am. J. Respir. Cell. Mol. Biol. 7, 317. Schoeffter, P., C. Lugnier, F. Demesy-Waeldele and J.C. Stoclet, 1987, Role of cyclic AMP- and cyclic GMP-phoshodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta, Biochem. Pharmacol. 36, 3965. Schoeffter, P. and J.C. Stoclet, 1985, Effect of vasoactive intestinal polypeptide (VIP) on cyclic AMP level and relaxation in rat isolated aorta, Eur. J. Pharmacol. 109, 275. Seamon, K.B. and J.W. Daly, 1983, Forskolin, cyclic AMP and cellular physiology, Trends Pharmacol. Sci. 4, 120. Shaul, P.W., K.M. Muntz and L.M. Buja, 1990, Comparison of /3 adrenergic receptor binding characteristics and coupling to adenylate cyclase in rat pulmonary artery versus aorta, J. Pharmacol. Exp. Ther. 252, 86. Sibley, D.R., P. Nambi, J.R. Peters and Lefkowitz, 1984, Phorbol diesters promote /3-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes, Biochem. Biophys. Res. Commun. 121,973. Singer, H.A., 1990, Phorbol ester-induced stress and myosin light chain phosphorylation in swine carotid medial smooth muscle, J. Pharmacol. Exp. Ther. 252, 1068. Toews, M.L., M. Liang and J.P. Perkins, 1987, Agonists and phorbol esters desensitize /3-adrenergic receptors by different mechanisms, Mol. Pharmacol. 32, 737. Weishaar, R.F., S.D. Burrows, D.C. Kobylakz, M.M. Quade and D.B. Evans, 1986, Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets, Biochem. Pharmacol. 35,787. Winder, S.J. and M.P. Walsh, 1990, Smooth muscle calponin. Inhibition of actomyosin Mg ATPase and regulation by phosphorylation, J. Biol. Chem. 265, 10148.
191
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53373
Effect of vascular smooth muscle relaxants on the protein kinase C-mediated contraction in the rat pulmonary artery J e a n - P i e r r e S a v i n e a u *, P a t r i c k G o n z a l e z D e L a F u e n t e a n d R o g e r M a r t h a n Laboratoire de Physiologie, Universit~ de Bordeaux II, 146 Rue L~o Saignat, 33076 Bordeaux Cedex, France
Received 19 April 1993, revised MS received 29 July 1993, accepted 24 August 1993
In the rat pulmonary artery, phorbol 12,13-dibutyrate induces a contraction due to the activation of the protein kinase C. We investigated the sensitivity of this protein kinase C-mediated contraction to a variety of vascular smooth muscle relaxants. Pretreatment of rat pulmonary artery with relaxant compounds altered the subsequent concentration-response curve to phorbol 12,13-dibutyrate (0.05-2/xM) in a variable manner. Isoprenaline (0.1-10/zM), nifedipine (0.01-1 /zM) and cromakalim (0.1-10 /zM) had no effect, whereas vasoactive intestinal peptide (VIP, 1-10 nM), forskolin (0.1-2 /xM), theophylline (0.1-2.5 mM), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20-1724, 2-20 /zM), dipyridamole (10-100 /xM), 8 bromo-cyclic GMP (8-br-cGMP, 5-500/zM) and dibutyryl cyclic AMP (db-cAMP, 100-500/~M) shifted the concentration-response curve to phorbol 12,13-dibutyrate to the right and decreased the maximal response. When cumulative concentrations of relaxants were applied on the plateau of the contraction induced by 0.2 or 2/xM phorbol 12,13-dibutyrate, again, isoprenaline, nifedipine and cromakalim failed to decrease the protein kinase C-mediated contraction, whereas the other agents produced concentration-dependent relaxation. From their inhibitory effect on the 0.2/zM phorbol 12,13-dibutyrate-induced contraction, the rank order of potency of these relaxants was: VIP >> forskolin > RO 20-1724 > 8-br-cGMP > theophylline > dipyridamole > db-cAMP. In chemically (fl escin) skinned preparations, cGMP (5-500/zM) and cAMP (50-1000/xM) antagonized in a concentration dependent manner the contraction induced by phorbol 12,13-dibutyrate at constant Ca z÷ concentration. These results show that protein kinase C-mediated contraction in pulmonary vascular smooth muscle is not altered by ion channel modulators or by stimulation of the fl-adrenoceptor with isoprenaline. On the other hand, protein kinase C-mediated contraction is reduced by other compounds which increase the intracellular cyclic nucleotide content. The interaction between protein kinase C and cyclic nucleotide-dependent protein kinases may be important in the physiological and pathophysiological control of tone in the pulmonary circulation. Pulmonary artery; Smooth muscle; Vascular relaxant; Phorbol ester; Protein kinase C; Phosphodiesterase inhibitor; Cyclic nucleotides
I. Introduction Activation of protein kinase C, an ubiquitous cellular enzyme (Nishizuka, 1986), may play a crucial role in the maintenance of tension in vascular smooth muscles stimulated by agonists (Rasmussen et al., 1987; Jiang and Morgan, 1987; Khalil and Morgan, 1992). This hypothesis is currently investigated using the tumour promoting phorbol esters which, easily, penetrate smooth muscles cells, bind to and activate protein kinase C (Kikkawa et al., 1983; Leach and Blumberg, 1989). Phorbol esters induce slow developing and sustained contraction in smooth muscles isolated from a variety of vascular beds (e.g. aorta, Jiang and Morgan, 1987; coronary arteries, Mori et aL, 1990; cerebral
* Corresponding author. Tel. 57 57 13 60, fax 56 99 03 80.
arteries, Salaices et al., 1990; saphenous vein, Chiu et al., 1988). Such is also the case in the pulmonary artery (Orton et al., 1990; Savineau et al., 1991). In the pulmonary circulation, protein kinase C has also been implicated in pathophysiological processes such as pulmonary hypertension (Marche, 1989; Dempsey et al., 1991) and hypoxic vasoconstriction (Orton et al., 1990; Jin et al., 1992). If, at least a part of these processes is linked to an abnormal activation of protein kinase C, it would be relevant to investigate the effect of smooth muscle relaxants on the protein kinase C-mediated contraction in the pulmonary circulation. The interaction between some relaxants and protein kinase C has been previously studied in the guinea-pig lung parenchymal strip (Obianime et al., 1989) which contains distal airway, microvascular and other contractile cells. However, direct investigation of this interaction in the isolated pulmonary artery has not yet been performed.
192
We therefore designed the present study to investigate the effect of ten vascular relaxants on the contractile response elicited by phorbol 12,13-dibutyrate, a potent protein kinase C-activator (Castagna et al., 1982) in the main pulmonary artery of the rat (Savineau et al., 1991). Relaxants were chosen to act via various pathways in the relaxation process. We thus have studied responses to ion channel modulators (nifedipine, cromakalim), membrane receptor agonists (isoprenaline, vasoactive intestinal peptide (VIP), an adenylate cyclase activator (forskolin), phosphodiesterase inhibitors (theophylline, 4-(3-butoxy-4-methoxybenzyl)-2imidazolidinone (RO 20-1724), dipyrimadole) and cyclic nucleotide analogues (8-bromo-cyclic GMP (8-brcGMP), dibutyryl cyclic AMP (db-cAMP)). We also loocked at the effect of cyclic AMP (cAMP) and cyclicGMP (cGMP) on the phorbol 12,13-dibutyrate-induced contraction in chemically (/3 escin) skinned preparations.
2. Materials and methods
2.1. Tissue preparation Experiments were performed on strips (200-400 tzm in diameter, 8-10 mm in length) isolated from the main pulmonary artery of male Wistar rats aged from 10 to 15 weeks. Animals were anesthetized by intraperitoneal injection of 40 mg sodium pentobarbitone. Heart and lungs were removed en-bloc. The main pulmonary artery was then rapidly dissected and opened. Strips (two to three for each artery) were cut transversely.
2.2. Mechanical recording Isometric contraction was measured in an experimental chamber (Savineau et al., 1988) by means of a highly sensitive force transducer (Akers 801, Horten, Norway) connected to a micromanipulator allowing to stretch the strip to a length close to the optimal length determined in preliminary experiments.
2.3. Solutions 2.3.1. Solutions for intact tissues Physiological solutions had the following composition. Reference solution (mM): NaCl 118.4, KCl 4.7, CaC12 2.5, MgSO4 1.2, KHEPO 4 1.2, NaHCO 3 25, glucose 11; the solution was aerated with 5% CO 2 in 0 2. A K+-rich (80 mM) solution was prepared by substituting an equimolar amount of KC1 for NaC1.
2.3.2. Solutions for skinned tissues The main relaxing solution used in these experiments had the following composition (mM): KCI 85, MgCI 2 5, ATP (Na) 2 5, EGTA 10, Tris 20, maleic acid 20 brought to pH 7.1 at 25°C with KOH. Solutions with variable free Ca 2÷ concentration were also prepared by mixing appropriate amounts of standard Ca 2÷ and EGTA solutions. The free Ca 2÷ concentration was calculated using a computer programm adapted from that of Fabiato (1988). The ionic strength of the solution was standardized at 0.2 M by adjusting the KC1 concentration. 2.4. Experiments with intact tissues 2.4.1. Pretreatment of the tissues with relaxants on the phorbol 12,13-dibutyrate-induced response At the beginning of the experiments, K+-rich (80 mM) solution was repeatedly applied in order to obtain at least three contractions similar in both amplitude and kinetics before the addition of phorbol 12,13-dibutyrate. A control concentration-response curve to phorbol 12,13-dibutyrate was constructed by adding cumulative concentrations of phorbol 12,13-dibutyrate (0.05-2 tzM). A concentration increment was made once the maximal contractile effect of the preceding concentration had been recorded (generally 10-12 min). A second then a third cumulative concentrationresponse curve to phorbol 12,13-dibutyrate was constructed in the presence of two different concentrations of one relaxant, applied for 15 min previously. It was verified, in control experiments, that three or even four identical concentration-response curves to phorbol 12,13-dibutyrate could be obtained from the same arterial strip provided that a time interval of 1 h was allowed between two successive cumulative concentration-response curves. The response to each concentration of phorbol 12,13-dibutyrate alone or of phorbol 12,13-dibutyrate in the presence of the relaxant was expressed as a percentage of the maximal response obtained in the first cumulative concentration-response curve.
2.4.2. Effect of relaxants on tissues precontracted with phorbol 12,13-dibutyrate In a second set of experiments, pulmonary artery strips were precontracted with 0.2 and 2 ~M phorbol 12,13-dibutyrate, concentrations inducing half-maximal and maximal responses, respectively as determined from the above experiments. When the contractile response plateaued, concentration-response curves to relaxants were constructed by adding cumulative concentrations of the compounds. A concentration increment was made once the maximal relaxant effect of the preceding concentration had been recorded: usually 8-10 min with forskolin or theophylline, and 15-18
193 min with isoprenaline, VIP, RO 20-1724, dipyridamole, db-cAMP, 8-br-cGMP, nifedipine or cromakalim. It was verified, in control experiments, that the contraction induced by phorbol 12,13-dibutyrate alone was maintained as long as 1 h without significant variation in amplitude (1.7 + 0.2% decrease in the amplitude of 0.2 txM phorbol 12,13-dibutyrate-induced contraction between 15 and 60 min, n --- 7, P > 0.05). The response to each concentration of relaxant was expressed as a percentage of the contractile response elicited by 0.2 or 2 /zM phorbol 12,13-dibutyrate alone. A mean curve was constructed which enabled the concentration producing a decrease of 50% of the phorbol 12,13-dibutyrate-induced contraction to be determined. Since it has been previously shown that phorbol 12,13-dibutyrate-induced contraction is endothelium independent (Savineau et al., 1991), experiments with these tissues were performed on endothelium intact pulmonary artery strips at 37 + 0.5°C.
were more consistent in amplitude when temperature was decreased below 30°C.
2.5. Experiments with /3 escin-treated tissues
2. 7. Analysis of results
Arterial strips used in these experiments were dissected as mentioned above and in addition were cleaned of their periarterial connective tissue and scratched on the luminal surface to remove the endothelium. After recording the contractile response to the K+-rich (80 raM) solution, the strip was incubated in the main relaxing solution in the presence of 80/~M /3 escin for 30 rain. During the 15 latter min of this skinning period, the ionophore A23187 (10 /xM) was also added to impair the Ca 2÷ accumulating and releasing functions of the sarcoplasmic reticulum. After removal of/3 escin and A23187 the success of skinning was attested by recording a contraction to 20/xM free Ca 2+ ions the amplitude of which was higher (115 + 10.4%) than that induced by K ÷ rich (80 mM) solution before the skinning procedure. Then, after complete relaxation, the Ca 2÷ concentration was adjusted to 0.08 /xM (pCa 7.1). Phorbol 12,13-dibutyrate (0.2 or 2 ~M) was applied inducing a maintained contraction. At the plateau of the response, a concentration-response curve to cAMP or cGMP was performed by adding cumulative concentration of the compounds. One concentration response curve was established for each /3 escintreated strip. The effect of the ionophore A23187 on the functioning of the sarcoplasmic reticulum was checked by comparing the contractile effect of inositol 1,4,5-trisphosphate in tissues exposed or unexposed to A23187. After the skinning procedure, the EGTA concentration was lowered to 0.1 mM and 0.5 /xM free Ca 2+ were applied for 8 min to load the sarcoplasmic reticulum. 10/zM inositol 1,4,5-trisphosphate was then added to the solution. Experiments with/3 escin-treated tissues were done at 25°C, since we observed, in preliminary experiments, that CaZ+-induced contractions
The amplitude of contractions is expressed as mean + S.D. for n, number of experiments. Significance was assessed by paired Student's t-tests. A difference between means was considered significant when P < 0.05.
2.6. Chemicals Chemicals used in the present study were: cAMP, db-cAMP, cGMP, 8-br-cGMP, dipyridamole, forskolin, inositol 1,4,5-trisphophate, isoprenaline, phorbol 12,13-dibutyrate, theophylline, VIP all from Sigma (La Verpill~re, France); RO 20-1724 and nifedipine from Gibco BRL (Cergy Pontoise, France); cromakalim was a gift from Smith Kline Beecham Research pharmaceuticals (Betchworth, U.K.). Dipyridamole, forskolin, nifedipine and phorbol 12,13-dibutyrate were dissolved in dimethylsulfoxide (DMSO), and cromakalim was dissolved in 70% ethanol. The maximal concentration of DMSO or ethanol in the bath was < 0.1% and had no effect on the contractility of main pulmonary artery strips.
3. Results
3.1. Effect of pretreatment of tissues with relaxants on the cumulative concentration-response curve to phorbol 12,13-dibutyrate Fig. 1 shows examples of the effect, on the pulmonary arterial tone, of cumulatively applied concentrations of phorbol 12,13-dibutyrate (0.05-2 ~M) alone (ABC, a) and of phorbol 12,13-dibutyrate in the presence of isoprenaline (10 /~M, Ab), theophylline (500 tzM, Bb) and db-cAMP (500 /xM, Cb). The results obtained with the ten relaxants tested are shown on figs. 2-4. VIP (1-10 nM), forskolin (0.1-2 /zM), RO 20-1724 (2-20 /zM), dipyridamole (10-100 /~M) theophylline (0.1-2.5 mM), 8-br-cGMP (5-500 /zM) and db-cAMP (100-500/zM) all induced a rightward shift of the cumulative concentration-response curve with a decrease in the maximal response (figs. 2, 3). For example, 2 IzM forskolin or 500/~M theophylline - the two most efficacious compounds - reduced 2 /~M phorbol 12,13-dibutyrate-induced contraction by 88.2 + 3.7% (n = 5) and 80.6 + 6.1% (n = 5), respectively, whereas 10 nM VIP reduced this contraction by only 36 + 7.8% (n = 5). Higher of concentrations VIP (50 and 100 nM) had 'no further effect on the cumulative concentration-response curve to phorbol 12,13-dibutyrate (n = 6, not shown). On the contrary, the cu-
194 A 5O
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Fig. 1. Original traces showing the effect of three vascular relaxants on contractions induced by phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentrations (0.05-2 ~ M ) of phorbol 12,13-dibutyrate (PDB) were applied in control tissues (ABCa) and in tissues pretreated with isoprenaline (10 p.M, Ab), theophylline (500/~M, Bd) and db-cAMP (500 /.LM, Cb). Relaxants were applied 15 min before and throughout the concentration-response curve for phorbol 12,13-dibutyrate.
mulative concentration-response curve to phorbol 12,13-dibutyrate was nearly unaltered in the presence of isoprenaline (0.1-10/xM, fig. 2A), nifedipine (0.01-1 /zM, fig. 4A) or cromakalim (0.1-10/zM, fig. 4B).
3.2. Effect of relaxants on tissues precontracted with phorbol 12,13-dibutyrate Main pulmonary artery strips stimulated with 0.2 /zM (fig. 5A) or 2 /zM phorbol 12,13-dibutyrate developed a contractile response which reached a plateau in 15 + 3.2 min ( n = 12) and 18.5 + 2.8 min ( n = 10), respectively, and could be maintained without significant change in amplitude for as long as 1 h. Removal of TABLE 1 Concentrations (~.M) of relaxants producing 50% decrease in the 0.2 and 2 p.M phorbol 12,13-dibutyrate-induced contractions. ( - ) = not determined. Values were determined from a m e a n curve obtained from five experiments for each relaxant. Relaxant
Isoprenaline VIP Forskolin RO-201724 Dipyridamole Theophylline 8-br-cGMP db-cAMP Nifedipine Cromakalim
Phorbol 12,13-dibutyrate 0.2/zM
2 ~M
0.0065 0.5 1.1 120 85 15 320 -
0.6 1.8 210 350 28 420 -
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, 8
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7
-log PDB (M)
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6
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7
6
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Fig. 2. Effect of cyclic nucleotide-elevating c o m p o u n d s on the cumulative concentration-response curve for phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentration-response curve was obtained from control tissues (e) and from tissues pretreated with: (A) isoprenaline (10/zM, • ) ; (B) VIP (1 nM * , 10 nM II); (C) forskolin (0.1 /zM • , 1 / z M II, 2 / ~ M o); (D) theophylline (100/~M • , 250/xM III, 5 0 0 / z M o); (E) R O 20-1724 ( 2 / x M • , 10 /zM II, 20 /xM o ) and (F) dipyridamole (10 • , 50 II, 100/~M o). In each panel, abscissa scale: - log concentration (M) of phorbol 12,13-dibutyrate (PDB) and ordinate scale: tension as a percentage of the maximal response induced by 2 /zM phorbol 12,13-dibutyrate in control tissues. Data points are mean, n = 5. Vertical lines show S.D. * indicates a response in the presence of relaxant which is significantly different ( P < 0.05) from the control experiment.
phorbol 12,13-dibutyrate induced a slow recovery of the basal tone which re-established in 25 + 3.2 min (n = 12) and 41 + 4.5 min (n --- 10) for 0.2 and 2 /zM phorbol 12,13-dibutyrate, respectively. Fig. 5BC shows an example of the effect of cumulatively applied concentrations of 8-br-cGMP and of 10 /zM cromakalim, respectively on the 0.2 /zM phorbol 12,13-dibutyrateinduced contraction. Concentrations of relaxants producing a 50% decrease in the contraction induced by 0.2 and 2 /xM phorbol 12,13-dibutyrate are listed in table 1. From their respective effect on tissues precontrated with 0.2 /zM phorbol 12,13-dibutyrate, the rank
195
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Fig. 3. Effect of cyclic nucleotide analogues on the cumulative concentration-response curve for phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentration-response curve was obtained from control tissues (e) and from tissues pretreated with: (A) db-cAMP (100 tzM • , 250 p.M I , 500 /zM ©) and (B) 8-br-cGMP (10 p.M • , 100 tzM I ) . In each panel, abscissa scale: - l o g concentration (M) of phorbol 12,13-dibutyrate (PDB) and ordinate scale: tension as a percentage of the maximal response induced by 2 /zM phorbol 12,13-dibutyrate in control tissues. Data points are mean, n = 5. Vertical lines show S.D. * indicates a response in the presence of analogue which is significantly different from the corresponding response in the control experiment.
Fig. 5. Effect of vascular relaxants on the rat isolated pulmonary artery pre-contracted with phorbol 12,13-dibutyrate. (A) Control contraction induced by 0.2/xM phorbol 12,13-dibutyrate (PDB). (B) Effect of cumulatively applied concentrations of 8-br-cGMP. (C) Effect of 10/zM cromakalim applied for the time indicated by the dotted line.
order of potency of relaxants appeared to be: VIP >> forskolin > RO 20-1724 > 8-br-cGMP > theophylline > dipyridamole > db-cAMP. Again in these experiments, isoprenaline (0.1-10 /~M, n =5), nifedipine (0.1-5 /zM, n = 4) and cromakalim (1-10 IzM, n = 4) failed to relax phorbol 12,13-dibutyrate-precontracted pulmonary artery. However, in similar experiments, 1 /.~M isoprenaline produced 50.3 + 4.2% (n = 5) relaxation in 80 mM KCl-precontracted tissues and propranolol (5 ~M) abolished this effect (data not shown).
tractile response, application of 0.2 (fig. 6Ba) or 2/xM phorbol 12,13-dibutyrate induced a sustained contraction the amplitude of which was 54.3 + 6.8 and 122 + 14.2% (n = 4) respectively of that produced by 20/xM Ca 2÷. The amplitude and kinetics of the phorbol 12,13-dibutyrate-induced contraction was not modified in arterial strips unexposed to the ionophore A23187 (fig. 6Aa). On the other hand, the contractile response to inositol 1,4,5-trisphosphate (10 ~M) was dependent on the pretreatment of the strip with the ionophore
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Fig. 4. Effect of ion channel modulators on the cumulative concentration-response curve for phorbol 12,13-dibutyrate in the rat isolated pulmonary artery. Cumulative concentration-response curve was obtained from control tissues (e) and from tissues pretreated with: (A) nifedipine (1 /zM, • ) and (B) cromakalim (10 g,M, • ) . In each panel, abscissa scale: - l o g concentration (M) of phorbol 12,13-dibutyrate (PDB) and ordinate scale: tension as a percentage of the maximal response induced by 2 /zM phorbol 12,13-dibutyrate in control tissues. Data points are mean, n = 5. Vertical lines show S.D.
2min
b
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Fig. 6. Effect of pretreatment with ionophore A23187 on the contraction induced by phorbol 12,13-dibutyrate and inositol 1,4,5-trisphosphate in fl escin skinned tissues. In tissues unexposed to A23187 (top panel A), phorbol 12,13-dibutyrate (PDB) and inositol 1,4,5-trisphosphate (IP 3) induce a contraction (Aa and Ab, respectively). In tissues exposed to A23187 (10/zM) applied during the last 15 min of the skinning procedure (lower panel B), contraction to phorbol 12,13-dibutyrate is unchanged (Ba) whereas contraction to inositol 1,4,5-trisphospbate is abolished (Bb). Prior stimulation with inositol 1,4,5-trisphosphate, the sarcoplasmic reticulum was loaded with 0.5 /zM Ca 2÷ for 8 rain. This result is typical of four experiments.
196 B
A 100
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/
6
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t
t
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Fig. 7. Effect of cyclic nucleotides on the phorbol 12,13-dibutyrateinduced contraction in /3 escin-treated preparations. Concentrationresponse curves for the effect of cGMP (A) and cAMP (B) on contractions induced by 0.2 (e) and 2 p.M ( a ) phorbol 12,13-dibutyrate, respectively. For each panel, abscissa scale shows - l o g concentration (M) of cyclic nucleotide and ordinate scale gives tension as a percentage of the phorbol 12,13-dibutyrate-induced contraction in the absence of cyclic nucleotide. Data points are mean, n = 4. Vertical lines show S.D. The free Ca 2÷ concentration was kept constant at 0.08/zM thoughout the experiments.
A23187 (fig. 6ABb). Further addition of cGMP (1-500 /xM) or cAMP (10-1000 /zM) at the plateau phase of the phorbol 12,13-dibutyrate response induced a concentration-dependent inhibition of this contraction (fig. 7). The concentrations producing 50% decrease of the 0.2 and 2 jzM phorbol 12,13-dibutyrate-induced contraction were 12 and 30 ~M cGMP; 120 and 210 ~M cAMP, respectively.
4. Discussion
Our study shows that the alteration in phorbol 12,13-dibutyrate-induced contraction in rat main pulmonary artery depends on the type of smooth muscle relaxants used. The phorbol 12,13-dibutyrate-induced contraction is antagonized by adenylate cyclase activator (forskolin), cyclic nucleotide phosphodiesterase inhibitors (theophylline, RO 20-1724, dipyridamole) and cyclic nucleotide analogues (db-cAMP, 8-br-cGMP), whereas it is not modified by ion channel modulators (nifedipine, cromakalim). Finally, relaxants acting at membrane receptors (isoprenaline, VIP) exhibit peculiar effect according to the type of the stimulated receptor. Previous studies (Orton et al., 1990; Savineau et al., 1991) have shown that phorbol 12,13-dibutyrate-induced contraction in the rat pulmonary artery is due to the activation of protein kinase C and is independent of both Ca 2÷ influx and Ca 2÷ release from internal stores. The fact that the Ca 2+ channel antagonist nifedipine and the potassium channel opener cromakalim (fig. 4) did not alter phorbol 12,13-dibutyrateinduced contraction whereas they inhibited KCl-induced contraction in this same preparation (Orton et al., 1990; Savineau and Marthan, 1993) corroborates
the fact that protein kinase C-mediated contraction is a membrane potential-independent process. Moreover, the fact that phorbol 12,13-dibutyrate-induced contraction in skinned tissues is similar whether the sarcoplasmic reticulum is functional or not (fig. 6) confirms that it is independent of Ca 2÷ release from internal stores. These findings suggest that the phorbol 12,13-dibutyrate-stimulated enzyme is a Ca-independent isoform of protein kinase C as also shown in the rat aorta (Collins et al., 1992). The main result of the present study is that protein kinase C-mediated contraction in the pulmonary circulation is sensitive to substances which increase or mimick intracellular cyclic nucleotides at a site distal to the membrane receptor. Among these relaxants, forskolin an activator of the catalytic subunit of adenylate cyclase (Seamon and Daly, 1983) appears the most potent (fig. 2C and table 1). This result is similar to those of Obianime and Dale (1989) and of Salaices et al. (1990), in rat aorta and cat cerebral arteries, respectively. Theophylline, a non-specific phosphodiesterase inhibitor, increases both the cAMP and cGMP content of smooth muscle cells (Weishaar et al., 1986) while RO 20-1724 and dipyridamole are specific inhibitors of cAMP-dependent and cGMP-dependent phosphodiesterase, respectively (Schoeffter et al., 1987; Ahn et al., 1992). Since the detailed mechanism by which protein kinase C contracts vascular smooth muscle is not yet fully elucidated, it is difficult to explain the mode of action of cyclic nucleotides on this protein kinase Cmediated contraction. Protein kinase C has been shown to phosphorylate a variety of proteins such as myosin light chains (Nishikawa et al., 1983; Singer, 1990), caldesmon (Adam et al., 1989), calponin (Winder and Walsh, 1990) and the myristorylated alanine rich C kinase substrate (Hartwig et al., 1992) which are involved in the control of the smooth muscle contractile apparatus. How cyclic nucleotides interact with these proteins is a matter of debate. However, the effect of cAMP and cGMP on the phorbol 12,13-dibutyrate-induced contraction in /3 escin-treated tissues (fig. 7) suggests that cAMP and cGMP-dependent protein kinases decrease whereas protein kinase C increases the sensitivity of the contractile machinery in pulmonary artery as previously shown in other vascular beds (Jiang and Morgan, 1987; Itoh et al., 1985; Nishimura and Van Breemen, 1989). Thus a direct effect of these kinases on the contractile apparatus could be the molecular basis for the interaction and the regulatory action of protein kinases on vascular smooth muscle tone. A paradoxical result of our study is the absence of effect of isoprenaline, a specific /3-adrenoceptor agonist, on phorbol 12,13-dibutyrate-induced contraction. This lack of effect cannot be ascribed to the absence or the non-functioning of/3-adrenoceptors in the rat main
197
pulmonary artery since it has been shown that the /3-adrenoceptor density in pulmonary artery is higher than in aorta (Shaul et al., 1990) and similar to that in the bronchial smooth muscle (Schell et al., 1992). Moreover isoprenaline relaxes KCl-precontracted pulmonary artery (O'Donell and Wanstall, 1984; present study). On the other hand, the coupling between the increase in cyclic AMP content and fl-adrenoceptor ligand binding (Shaul et al., 1990) is reduced by phorbol 12,13-dibutyrate which decreases by approximately 50% the cyclic AMP production in response to isoprenaline without altering its Bmax (Aiyar et al., 1987). Phorbol ester-activated protein kinase C phosphorylates /3-adrenoceptors (Sibley et al., 1984), thus inducing an heterologous desensitization without internalization of these receptors (Aiyar et al., 1987; Toews et al., 1988). The resulted uncoupling between /3-adrenoceptors and adenylate cyclase could be responsible for the insensitivity to isoprenaline of the protein kinase Cmediated contraction in the rat main pulmonary artery as also observed in the rat aorta (Obianime and Dale, 1989). The other receptor agonist used in the present study was VIP, a transmitter of the non-adrenergic noncholinergic system in the pulmonary tract (Barnes, 1988). VIP receptors have been localized on the smooth muscle membrane of pulmonary vessels (Dey et al., 1981; Laitinen et al., 1985), and VIP is a potent relaxant of isolated pulmonary arteries precontracted with prostaglandin H 2 (Hamasaki et al., 1983). The potency of VIP on the protein kinase C-mediated contraction was high (in the nM range) (table 1) but its efficacy (fig. 2B) was limited even for the highest tested concentration (100 riM). This limited efficacy could be explained by the fact that the mechanism underlying VIP action is only partly related to an increase in cAMP content (Schoeffter and Stoclet, 1985) or/and by a desensitization of the receptor similar to that of /3-adrenoceptors. In conclusion, our study has shown that protein kinase C-mediated vasoconstriction in the pulmonary circulation can be reversed by substances which by-pass the receptor agonist step and bring about an increase in the cyclic nucleotide content of vascular smooth muscle cells. Thus, interactions between protein kinase C and cyclic nucleotide-dependent protein kinases may play a role in the control of pulmonary vascular tone. Whether these interactions would be altered in pathological processes involving the pulmonary circulation merits further investigations. Acknowledgements The authors thank Ms H. Crevel for technical assistance and Ms M.C. Chauvet for typing the manuscript. This work was supported by a grant from 'Conseil R6gional d'Aquitaine' (no. 9203023).
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