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

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Cellular Signalling Vol. 7, No. 6, pp. 571-581, 1995. Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0898-6568/95 $9,50 + 0.00

Pergamon 0898-6568(95)00026-7

INHIBITORY

EFFECT

PHOSPHOINOSITIDE CULTURED

OF PHORBOL HYDROLYSIS

CANINE

ESTER ON BRADYKININ-INDUCED AND

TRACHEAL

CALCIUM

SMOOTH

MOBILIZATION

MUSCLE

IN

CELLS

SHUE-FEN LUO,* HUI-LIANG TSAO,t RICHARD O N G , t JEN-TSUNG H S I E H t and CHUEN MAO YANGt~ * Internal Medicine and Cellular and Molecular Pharmacology Laboratory and Department of I"Pharmacology, Chang Gung College of Medicine and Technology, 259 Wen-Hwa 1 Road, Kwei-San, Tao-Yuan, Taiwan, Republic of China (Received 15 February 1995)

Abstract--Regulation of the increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca 2+ concentration ([Ca2+]0 by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by bradykinin (BK) led to IP3 formation and caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 IxM) for 30 min blocked the BK-induced IP3 formation and Ca2÷ mobilization. However, this inhibition was reduced after incubating the cells for 4 h with PMA. Inactive phorbol ester, 4a-phorbol 12,13-didecanoate at 1 IxM, did not inhibit these responses to BK. Prior treatment with stanrosporine (1 ~tM), a PKC inhibitor, inhibited the effect of PMA on the BK-induced response, suggesting that the effect of PMA is mediated by the activation of PKC. In parallel experiments, a change of PKC activity was observed. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the cell membranes within 30 rain. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Moreover, treatment with 1 uM PMA for 2 and 24 h did not significantly change the Ko and Bmaxof the BK receptor for [aH]BK binding (control: Ko = 2.3 _+ 0.3 nM, Bm~x= 25.2 + 1.4 fmol/mg protein). These results suggest that activation of PKC inhibit IP3 accumulation and consequently attenuate [Ca2+]i increase or inhibit independently both responses. The PMA-induced inhibition of responses to BK was associated with an increase in membranous PKC activity. Key words: Phorbol ester, Inositol phosphates, Ca2+, Bradykinin, Protein kinase C, Tracheal smooth muscle ceils.

INTRODUCTION

and subsequently an increase in intracellular free calcium ([Ca2+]i) [1]. In many cell types, including the neuroblastoma-glioma hybrid NG 108-15 [2] and h u m a n astrocytoma cell line D 384 [3], bradykinin (BK) receptors activate phospholipase C (PLC)-mediated PI hydrolysis in the plasma membrane. The resultant increase in IPa releases Ca 2+ from internal stores in several types o f cells including the NG 108-15 [2], endothelial cells [4], and NCB-20 ceils [5]. In tracheal smooth muscle, BK has been known to stimulate IP3 accumulation in the bovine [6] and canine [7],

It is well established that many agonists cause contraction o f tracheal smooth muscle through the activation of phosphoinositide (PI) hydrolysis to generate inositol 1,4,5-trisphosphate (IP3)

~/Authorto whomall correspondenceshouldbeaddressed. Abbreviations: BK- bradykinin; DAG- diacylglycerol; IP3--inositol 1,4,5,-trisphosphate; PI-phosphoinositide; PKC-protein kinase C; PLC- phospholipase C; PMAphorbo112-myristate 13-acetate; TSMCs-- tracheal smooth muscle cells.

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and subsequently increase in [Ca2+]i [8, 9]. Thus, in terms of second messenger generation, contraction of tracheal smooth muscle may be mediated by IP3-induced Ca 2÷ mobilization from internal stores. Activation o f PI hydrolysis by agonists also leads to the formation o f diacylglycerol (DAG), which activates the important regulatory enzyme protein kinase C (PKC) [10]. The P I - C a 2+ signalling system has been reported to be negatively regulated by PKC activation through a decrease in PI hydrolysis in several cell types [11-13], intracellular Ca 2÷ mobilization [14, 15], or both responses [2, 16]. These reports suggest that there are many candidates for target sites of PKC in the regulation of PI-Ca 2÷ signalling transduction in these cells. Phorbol esters, which mimic the effect of D A G and cause permanent activation o f PKC [17], are useful to examine the role o f D A G in the regulation of PI hydrolysis and Ca 2+ mobilization related to the change in PKC activity. Furthermore, phorbol ester has been shown to inhibit carbachol- and histamine-induced IP3 formation and calcium mobilization in cultured TSMCs [18-20]. Therefore, a question arises as to whether phorbol 12-myristate 13-acetate (PMA) exerts an inhibitory or a stimulatory effect on BK-induced responses in canine TSMCs. The precise molecular and biochemical mechanisms of phorbol esters related to desensitization o f BK-mediated physiological responses in tracheal smooth muscle are not clear. The purpose o f the present study was to investigate the role of the D A G - P K C pathway in the modulation o f the pharmacological properties of BK receptors, P I breakdown, change in [Ca2÷ ]i, and the involvement o f PKC in the desensitization o f BK receptors by phorbol esters.

standard laboratory chow and tap water ad libitum. Dogs were anesthetized with pentobarbitone (30 mg/ Kg, intravenously) and the lungs were ventilated mechanically via an orotracheal tube. The tracheae were surgically removed. Isolation o f tracheal smooth muscle cells

The TSMCs were isolated according to methods previously reported [21]. The trachea was cut longitudinally through the cartilage rings and the smooth muscle was dissected. The muscle was minced and transferred to the dissociation medium containing 0.2070 collagenase IV, 0.01% deoxyribonuclease I, 0.01 °70elastase IV, and antibiotics (100 U/mL penicillin G, 100 lig/mL streptomycin, and 250 ng/ml fungizone) in physiological solution. The physiological solution contained (mM): 137 NaCI; 5 KCI; 1.1 CaC12; 20 NaHCO3; 1 NaH2PO4; 11 glucose and 25 HEPES (pH 7.4). The tissue pieces were gently agitated at 37°C in a rotary shaker for 1 h, The released cells were collected and the residuum was again digested with fresh enzyme solution for an additional h at 37°C. The released cells were washed twice with DMEM/F-12 medium (1:1 v/v). The cells, suspended in DMEM/ F-12 containing 10°70FBS, were plated onto a 60 mm culture dish and incubated at 37°C for 1 h to remove fibroblasts which attached to the dish more rapidly than TSMCs. The cell number was counted and the suspension was diluted with DMEM/F-12 t o 2 x 105 cells/mL. The cell suspension was plated onto (0.5 mL/well) 24-well, (1 mL/well) 12-well, or (2 mL/ well) 6-well culture plates containing glass coverslips coated with collagen for receptor binding assay, inositol phosphates (IPs) accumulation and Ca 2÷ measurement. The medium was changed after 24 h and then every 3 days. After a 5-day culture, cells were changed t o DMEM/F-12 containing 1070FBS for 24 h at 37 °C. Then, the cells were incubated in DMEM/F-12 containing 107oFBS supplemented with insulin-like growth factor I (IGF-I, 10 ng/mL) and insulin (1 ~tg/mL) for 12-14 days. In order to characterize the isolated and cultured TSMCs and to exclude contamination by epithelial cells and fibroblasts, the cells were identified by an indirect immunofluorescent staining method using a monoclonal antibody recognition of smooth muscle light chain myosin [22]. Over 95070of the cell preparation was composed of smooth muscle cells.

MATERIALS AND METHODS Animals

Accumulation o f inositol phosphates

Mongrel dogs of either sex, 10-20 Kg, purchased from a local supplier, were used throughout this study. Dogs were housed in animal facilities under automatically controlled temperature and light cycle and fed

Effect of BK on the hydrolysis of PI was assayed by monitoring the accumulation of 3H-labelled inositol phosphates (IPs) as described by Berridge et al. [23]. Cultured TSMCs were incubated with 5 ;iCi/mL of

Phorbol ester and signal transduction myo-[2-3H] inositol at 37°C for 2 days. TSMCs were washed two times with PBS and incubated in KrebsHenseleit buffer (KHS, pH 7.4) containing (in mM) 117 NaCI, 4.7 KCI, 1.1 MgSO4, 1.2 KH2PO4, 20 NaHCO3, 2.4 CaCI2, 1 glucose, 20 HEPES, and 10 LiCI at 37°C for 30 min. After BK was added at the concentration indicated, incubation was continued for another 20 min. When phorbol esters were used, it was added at the time indicated before the addition o f BK. Reactions were terminated by addition of 5070 perchloric acid (PCA) followed by sonication and centrifugation at 3000 × g for 15 min. The P C A soluble supernatants were extracted four times with ether, neutralized with potassium hydroxide, and applied to a column of AG1-X8, formate form, 100-200 mesh (Bio-Rad). The resin was washed successively with 5 mL of water and 5 mL of 60 mM ammonium formate-5 mM sodium tetraborate to eliminate free myo- 1~H]inositol and glycerophosphoinositol, respectively. Sequential washes with 5 mL of 0.2 M ammonium formate/0.1 M formic acid, 0.4 M ammonium formate/0.1 M formic acid, and 1 M ammonium formate/0.1 M formic acid were used to elute inositol monophosphate (IP0, inositol bisphosphate (IP2), and inositol trisphosphate (IP3), respectively. The amount of [3H]IPs was determined in a radiospectrometer (Beckman TA5000). Measurement o f intracellular Ca 2+ level [ C a 2 + ]i was measured in confluent monolayers with the calcium-sensitive dye fura-2/AM as described by Grynkiewicz et al. [24]. Upon confluence, the cells were cultured in DMEM/F-12 with 1070FBS one day before measurements were made. The monolayers were covered with 1 ml of DMEM/F-12 with 1070FBS containing 5 lxM fura-2/AM and were incubated at 37°C for 45 min. A t the end of the period, the coverslips were washed twice with the physiological buffer solution containing (mM): 125 NaCI, 5 KCI, 1.8 CaC12, 2 MgCI2, 0.5 NaH2PO4, 5 NaHCO3, 10 HEPES, and 10 glucose, p H 7.4. The cells were incubated in PBS for further 30 min to complete dye de-esterification. The coverslip was inserted into a quartz cuvette at an angle of approximately 45 ° to the excitation beam and placed in the temperature controlled holder of an SLM 8000C spectrofluorometer (SLM Aminco, Urbana, IL, USA). Continuous stirring was achieved with a magnetic stirrer. Fluorescence o f Ca2÷-bound and unbound fura-2 was measured by rapidly alternating the dual excitation wavelengths between 340 and 380 nm and electronically separating the resultant fluorescence signals at emission wavelength 510 nm. The autofluorescence o f each monolayer was subtracted from the fluorescence data. The ratios (R) of the fluorescence at the

573

two wavelengths are computed and used to calculate changes in [Ca2+]i. The ratios of maximum (Rm,) and minimum (Rmm) fluorescence of fura-2 were determined by adding ionomycin (10 ~tM) in the presence of PBS containing 5 mM Ca 2÷ and by adding 5 mM E G T A at pH 8 in a Ca 2+-free PBS, respectively. The Ka of fura-2 for Ca 2+ was assumed to be 224 nM [24]. Protein kinase C assay Cultured TSMCs were washed with phosphatebuffered saline, and then sonicated for 1 min in 1 mL of Tris-HC1 buffer (20 mM, p H 7.5) containing 0.5 mM EGTA, 1 mM dithiothreitol, 2 mM benzamidine, 1 I~g/mL leupeptine and 0.1 lxg/mL pepstatine. The sonicate was centrifuged at 1000 × g for 8 min to remove unbroken cells and nuclei. Soluble and particulate fractions were separated by high speed centrifugation (100,000 x g for 1 h). Protein kinase C was assayed at 30°C as described [25]. The reaction mixture (250 ~tL) contained 20 mM PIPES buffer (pH 7.0), type IIIs histone (0.5 mg/mL), 0.1 mM dithiothreitol, 5 mM MgCI2, and 30 ~tg/mL of protein. The reaction was initiated by the addition of 25 IxM r-32p-ATP ( - 8000 cpm/~tL) and incubated at 37°C for 5 min. Additional components of the reaction mixture were: 0.1 mM EGTA, 25 Ixg/ml phospatidylserine, 1 ~tM PMA. Samples (150 Ixl) o f the reaction mixture were added to 4 mL of an ice-cold mixture of 507o trichloracetic acid. The diluted samples were filtered through 0.45 Ixm filters and washed eight times with 2 mL of the same solution without radioisotope and protein. Filters and samples (5 ~tL) of the incubation mixture were treated identically for scintillation counting. Enzyme levels were normalized to protein contents measures by the method of Bradford [26]. I~H]BK binding assay For detecting the effect of P M A on BK receptor density or affinity of TSMCs, [aH]BK was used as a ligand. Binding assays were performed with confluent TSMCs in 24-well culture plates, with or without PMA treatment in DMEM/F-12 containing 1070 FBS for either 2 or 24 h prior to the binding experiments, as described [27]. Culture medium was removed and 1 mL of binding buffer (20 mM HEPEs, p H 7.4, 17 mM NaC1, 5.4 mM KCI, 0.44 mM KH2PO4, 0.63 mM CaC12, 0.21 mM MgSO4, 0.34 mM Na2HPO4, 110 mM N-methylglucamine, 0.1 °70 (w/v) BSA and 2 mM bacitracin) was added to each well. Plates were equilibrated on ice for 10 min, after which the binding buffer was replaced with 0.25 mL of binding buffer containing the appropriate concentration of [3H]BK in the absence or presence of unlabelled BK (10 ~tM).

S.-F. Luo et al.

574

After 4 h incubation at 4°C, the binding buffer was removed and the cells were washed three times with 2 mL of binding buffer at 4°C. Cells were suspended in 0.25 mL of 0.1 NaOH and counted in a radiospectrometer. Counting data were corrected for the specific activity and quenching. The amount of specific binding was calculated as the total binding minus the binding in the presence of 10 ~tM unlabelled BK. Total receptor density (Bm~) and dissociation constant (KD) were calculated by Ligand program, as described previously [21].

1.1 ~ xl' I C)

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Analysis o f data The ECs0 values were estimated by Graph Pad Program and expressed as the mean (-logEC~o) + S.E. mean (Graph Pad, San Diego, California, U.S.A.). The concentration-effect curves were constructed by non-cumulative addition o f BK and fitted by sigmoid curve (log scale). The data were expressed as the mean + S.E. mean of experiments with statistical comparisons based on a two-tailed Student's t-test at a p < 0.01 level of significance.

Materials

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Dulbecco's modified Eagle's medium (DMEM)/ Ham's nutrient mixture F-12 (F-12) medium and foetal bovine serum (FBS) were purchased from J. R. Scientific (Woodland, CA). Insulin and insulin-like growth factor (IGF-I) were from Boehringer Mannheim (GmbH, Germany). Myo-[3H]inositol (18 C i / mmol), [3HIBK (68 Ci/mmol) and r-32p-ATP (3000 Ci/mmol) were from Amersham (Buckinghamshire, England). F u r a - 2 / A M was from Molecular Probes Inc. (Eugene, OR). Enzymes and other chemicals were from Sigma Co. (St. Louis, MO). RESULTS

Effects o f P M A on BK-stimulated IP~ accumulation In order to determine whether PKC activation b y P M A caused an inhibition o f BK-induced IP3 a c c u m u l a t i o n , the c o n c e n t r a t i o n - r e s p o n s e relationship for PMA inhibition of BK-stimulated I P , , IP2, a n d IP3 a c c u m u l a t i o n was e x a m i n e d in c u l t u r e d T S M C s . A s s h o w n in Fig. 1, signific a n t effects a r e o b t a i n e d with 10 n M P M A , well c o n s i s t e n t w i t h p r e v i o u s r e p o r t s in several cell t y p e s [7, 11-13, 18, 28]. T h e i n h i b i t o r y effect o f P M A o c c u r r e d in a c o n c e n t r a t i o n - d e p e n d e n t manner. PMA induced half-maximal inhibition

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log [PMA] (M) Fig. 1. Concentration dependence of P M A inhibition of BK-stimulated [3H]IPs accumulation. Cells prelabelled with [3H]inositol were washed with KHS, treated with various concentrations of P M A (0.1 n M 1 I~M) for 30 min and then exposed to BK (1 laM) for 20 min. The accumulation o f (0) IP 1, ( ' ) IP2, and ( • ) IP3 was determined, as described under "Methods." Values are expressed as the means +_ S.E. mean from four separate experiments determined in triplicate. ( - l o g E C s 0 ) o f B K - s t i m u l a t e d IP~, IP2 a n d IP3 f o r m a t i o n a t 8.7 _+ 0.4, 9.0 _+ 0.2, a n d 9.3 _+ 0.2 M , n = 4, respectively. It h a d n o effect o n the b a s a l levels o f I P s a c c u m u l a t i o n at a n y o f the c o n c e n t r a t i o n s tested. T h e i n h i b i t o r y a c t i o n o f P M A (10 n M ) was m a n i f e s t as a decrease in

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Fig. 3. Time course of PMA-induced inhibition of BK-stimulated [Ca2+]i change in cultured TSMCs. Cells were incubated in the absence (control, 0) or presence of 1 I~M P M A for various times. TSMCs grown on glass coverslips were loaded with 5 ~tM fura-2/AM and fluorescence measurement of [Ca 2÷]i was carried out in a dual excitation wavelength spectrofluorometer, with excitation at 340 and 380 nm, when 1 IxM BK was added. Values are expressed as the mean + S.E. mean from eight separate experiments. *p < 0.001 as compared with control.

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log [BDK] (M) Fig. 2. Effect of PMA on concentration-effect curves for BK-stimulated [3H]IPs accumulation in cultured TSMCs. Cells prelabelled with [3H]inositol were incubated in the absence (©) or presence of 10 nM P M A ( • ) for 30 min and then exposed to various concentrations of BK for 20 min. The accumulation of (a) IP~, (b), IP2, and (c) IP3 was determined, as described under "Methods." Values are expressed as the mean + S.E. mean from four separate experiments determined in triplicate.

the m a x i m a l r e s p o n s e , r a t h e r t h a n f r o m a shift to the right in the c o n c e n t r a t i o n - e f f e c t curve f o r B K - i n d u c e d IPs a c c u m u l a t i o n (Fig. 2). The h a l f m a x i m a l v a l u e s ( - IogECs0) f o r the s t i m u l a t o r y effect o f BK o n IP~, IP2, a n d IP3 f o r m a t i o n in

the p r e s e n c e o f 10 n M P M A (7.7 + 0.4, 7.3 _+ 0.5, a n d 7.1 + 0.4 M , n = 4, respectively) were close t o t h o s e o f c o n t r o l (7.4 + 0.5, 7.3 ___ 0.4, a n d 7.0 + 0.5 M, n = 4, respectively).

Effects o f PMA on the [Cae+]i response to BK A n increase in m e m b r a n o u s P K C activity is k n o w n to i n h i b i t I P s p r o d u c t i o n [11]. T h e effects o f BK, a c o n s t r i c t o r o f s m o o t h muscle t h a t operates through calcium-dependent pathways, was s t u d i e d o n [Ca2+]i. I n o r d e r t o e x a m i n e a p o s s i b l e role o f P K C in r e g u l a t i o n o f [Ca2+]i in c u l t u r e d T S M C s , we t e s t e d the effect o f P M A , a k n o w n a c t i v a t o r o f P K C , o n i n t r a c e l l u l a r C a 2+ t r a n s i e n t i n d u c e d b y BK. F i g u r e 3 shows t h e time course of PMA treatment on BK-induced changes in [Ca2+]i. P M A t r e a t m e n t f o r v a r i o u s t i m e p e r i o d s d i d n o t c h a n g e the resting level o f [Ca 2+ ]i. It d i d , h o w e v e r , s i g n i f i c a n t l y inhibit the t r a n s i e n t e l e v a t i o n o f [Ca2+]i i n d u c e d b y BK in T S M C s w h e n t r e a t e d with 1 ~tM P M A b e t w e e n 30 m i n a n d 2 h ( p < 0.001, as c o m p a r e d with

576

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Fig. 4. ~ncentrati~ndependence~fPMAinhibiti~n~fBK~stimu~ated[Ca2~]ichangeincu~turedTSMCs.TSMCs grown on glass coverslips were loaded with 5 ~tM fura-2/AM at 37°C for 45 min, washed with PBS, and incubated with various concentrations of PMA (a, 0.1 nM; b, 1 nM; c, 10 nM; d, 100 nM) for 30 min. Fluorescent measurement of [Ca2+]i was carried out as described under "Methods." BK (1 ~tM) was added at the indicated arrow. Values are expressed as the mean + S.E. mean from eight separate experiments.

control cells). Treatment with P M A for more than 4 h reduced its inhibitory effect, while [Ca 2÷]i response to BK after treatment o f T S M C s with P M A for 12 h showed the same extent as that o f the control cells (Fig. 3). This p h e n o m e n o n is well consistent with a previous report that long-term exposure of cells to phorbol esters causes down-regulation o f P K C and loss o f responsiveness to phorbol esters [30]. Figure 4A depicts tracings of BK-induced [Ca2+]i changes in TSMCs following treatment with P M A for 30 min. In control cells, the resting level of [Ca2+]i was 105 -+ 14 nM (n = 30). Addition o f BK (1 gM) resulted in a biphasic elevation of [Ca2+]i consisting o f a rapid, transient c o m p o n e n t at 306 + 18 nM within 30 s, and followed by a lower sustained component (194 -+ 15 nM, n = 8). P M A (0.1-1000 nM) did not significantly alter the resting level o f [Ca2+]i (107 _+ 4 nM). As shown in Fig. 4A, prior treatment of TSMCs with P M A followed by subsequent exposure to 1 I~M BK markedly inhibited the peak [Ca2+]i. Fig. 4B summarizes the effects o f varying concentrations of P M A on

BK-induced increase in [Ca2+]i. P M A induced half-maximal ( - logECs0) and maximal inhibition ( - l o g E C m 0 ) of BK-stimulated [Ca2+]i changes at 8.6_+ 0.3 and 6.0 M, (n = 8), respectively. The inhibitory action for P M A produced both depression o f the maximal responses and a shift to the right of the log the concentration-effect curve of BK (Fig. 5). The half maximal value ( - logECs0) for the stimulatory effect o f BK on Ca 2+ mobilization in the presence of 10 nM P M A (6.1 _+ 0.4 M) was higher than that of the control cells (7.1 _+ 0.3 M). Table 1 shows the effect of P M A on BKstimulated Ca 2+ release f r o m its intracellular stores and BK-induced Ca 2÷ influx. Pretreatment o f TSMCs with either 10 or 100 nM for 30 min inhibited the BK-stimulated C a 2+ release f r o m intracellular stores by 41 and 58070, respectively, while the BK-induced Ca 2+ influx was not significantly changed as compared with control.

Effect of staurosporine We examined the effect of staurosporine on PMA-induced inhibition of [Ca2+]i response to

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Fig. 5. Effect of PMA on concentration response curves for BK-induced [Ca2+]i change in cultured TSMCs. TSMCs grown on glass coverslips were loaded with 5 gM fura-2/AM at 37°C for 45 min, washed with PBS, and incubated in the absence (A) or presence (B) of 10 nM PMA at 37°C for 30 min. Fluorescent measurement of [Ca2+]i was carried out as described under "Methods," with various concentrations of BK (a, 10 I~M; b, 1 gM; c, 100 nM, d, 10 nM; e, 1 nM) added at the indicated arrow. Data expressed as the mean _+ S.E. mean from eight separate experiments are shown in (C). BK. As s h o w n in Fig. 6, T S M C s were treated with p h o r b o l esters (1 g M ) a n d t h e n s t i m u l a t e d with 1 lxM BK. T r e a t m e n t o f T S M C s with 1 IxM P M A for 30 m i n i n h i b i t e d the B K - s t i m u l a t e d [Ca2+]i r e s p o n s e b y 81010 (19 < 0.001, n = 8, as c o m p a r e d with the control). W h e n T S M C s were p r e i n c u b a t e d with s t a u r o s p o r i n e (1 ~tM), the inh i b i t o r y effect o f P M A o n B K - s t i m u l a t e d Ca2+ m o b i l i z a t i o n was reversed, a l t h o u g h pretreatm e n t with s t a u r o s p o r i n e a l o n e did n o t affect the [Ca2+]i response to BK. It is clear that the inhibition could be prevented by a P K C inhibitor. T h e inactive p h o r b o l ester, 4 a - p h o r b o l 12,13d i d e c a n o a t e ( 4 a - P D D , 1 lxM), did n o t block B K - i n d u c e d [Ca2+]i m o b i l i z a t i o n (Fig. 6). Table 1. Effect of PMA on BK-induced a rise in [Ca2+]i due to release from intracellular stores and Ca2+ influx in TSMCs Treatment Control PMA (10 nM) PMA (100 nM)

Ca2+ release (nM)

Ca2+ influx (nM)

65.1 + 4.7 38.2 + 3.0* 27.3 + 6.6*

21.1 _+ 2.6 22.6 + 6.3 18.3 + 2.1

TSMCs were preincubated with PMA (10 or 100 nM) or without (control) for 30 min. Cells were then stimulated with 1 gM BK in a Ca2+-freebuffer, to which 1.8 mM Ca2+ was added after completion of the BK-induced [Ca2÷]i from intracellular stores. Data are expressed as the mean + S.E. mean of six measurements. *p < 0.001 as compared with control.

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STA 4c~-PDD +

PMA Fig. 6. Effects of phorbol esters and staurosporine on BK-induced [Ca2+]i in cultured TSMCs. TSMCs grown on glass coverslips were loaded with 5 I~M fura-2/AM at 37 °C for 45 min, washed with PBS, and incubated with PMA (1 IxM), staurosporine (STA, 1 p.M), STA plus PMA, or 4a-PDD (1 ltM) at 37°C for 30 min. Fluorescent measurement of [Ca 2+]i was carried out as described under "Methods," with BK (1 ltM) added at the indicated arrow. Values are expressed at the mean _+ S.E. mean from eight separate experiments.

Time course o f PMA-induced PKC down-regulation A n i m p o r t a n t aspect o f the a c t i v a t i o n process o f P K C a p p e a r s to be the t r a n s l o c a t i o n o f the e n z y m e f r o m the cytosolic c o m p a r t m e n t to the

S.-F. Luo et al.

578

.-~ 2,5 •

E

O membrane

0

.~ 2.0

cytoso]ic

E O

Ec~ 1.5 1,0

O-

O

It

-~ i

(9

<

0,5

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0.0

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Time (hr) Fig. 7. Changes in PKC activity in canine tracheal smooth muscle ceils incubated with P M A for various time periods. Cultured cells were treated with P M A (1 tiM) for the indicated times. The cytosolic (O) and membraneous (O) fractions were assayed as described under "Methods." Data are expressed as mean _+ S.E. mean of four separate experiments determined in triplicate. p l a s m a m e m b r a n e [11]. P K C a c t i v i t y was m e a sured in b o t h t h e c y t o s o l i c a n d t h e p a r t i c u l a t e f r a c t i o n s o f canine T S M C s t h a t were either untreated or i n c u b a t e d with P M A (1 ~tM) for various p e r i o d s o f t i m e . F i g u r e 7 shows t h a t P M A t r e a t m e n t i n d u c e d a t r a n s i e n t increase in m e m b r a n e b o u n d P K C activity with a m a x i m u m o f 2.05 _+ 0.04 p m o l / m i n / m g p r o t e i n at 30 m i n (n = 4, p < 0.001, as c o m p a r e d with t h e u n t r e a t e d cells, 0.78 _+ 0.10 p m o l / m i n / m g p r o t e i n ) , d e c r e a s e d t o 0.94 + 0.02 p m o l / m i n / m g p r o t e i n a f t e r 2 h a n d p e r s i s t e d f o r at least 24 h o f P M A treatment. I n contrast, P M A i n d u c e d a sustained s u p p r e s sion o f P K C activity in t h e cytosolic f r a c t i o n .

f o r [3H]BK, c o m p l e t e s a t u r a t i o n b i n d i n g curves were o b t a i n e d f o r p a r a l l e l c o n t r o l cultures a n d cultures i n c u b a t e d with 1 ~tM P M A f o r either 2 o r 24 h ( T a b l e 2). S c a t c h a r d analysis s h o w e d t h a t c o n t r o l cells h a d a single class o f b i n d i n g sites with a Bmax o f 25.2 + 1.4 f m o l / m g p r o t e i n a n d a KD o f 2.3 + 0.3 n M . N o significant difference in the Bm~xa n d KD o f the BK r e c e p t o r s was o b t a i n e d w i t h cells t r e a t e d with P M A for 2 o r 24 h, as c o m p a r e d to c o n t r o l cells ( T a b l e 2).

Effect o f P M A on the density and affinity o f the B K receptors

Treatment

Table 2. Effect of PMA treatment on [3H]BK binding in cultured canine TSMCs (fmol/mg protein)

KD (nM)

25.2 +_ 1.4 26.3 + 1.5 24.7 + 1.7

2.3 +_ 0.3 2.6 _+ 0.4 2.7 _+ 0.4

Bm~

L o n g - t e r m i n c u b a t i o n with p h o r b o l esters has b e e n r e p o r t e d to cause a d o w n - r e g u l a t i o n o f a g o n i s t r e c e p t o r s in s o m e types o f cells [28, 29]. T o determine whether the observed P M A - i n d u c e d i n h i b i t i o n o f responses s t i m u l a t e d b y BK represented a n a c t u a l d e c r e a s e in the n u m b e r o f b i n d ing sites o r a c h a n g e in t h e KD o f the BK r e c e p t o r s

Control PMA (2 h) PMA (24 h)

Cultured TSMCs were incubated in the absence (control) or presence of PMA (1 t~M) for 2 and 24 h. Binding assays were performed in triplicate with concentrations of [3H]BK ranging from 0.2 to 10 nM and incubated at 4 ° C for 4 h. Data are expressed as the mean + S.E. mean of three individual experiments.

Phorbol ester and signal transduction DISCUSSION A rapid PI breakdown after receptor activation has been observed in several tissues in response to stimuli, such as neurotransmitters, growth factors, hormones, and light [31]. BKinduced hydrolysis o f PI, with the subsequent generation o f IP3 and DAG and the rise in [Ca 2+]i, can be attenuated by short-term activation of PKC in several cell types [2, 5]. These findings suggest that DAG formation during activation of BK receptors may feed-back to terminate phospholipase C (PLC)-mediated IP3 accumulation and Ca 2÷ mobilization. Furthermore, such a modulation of signal transduction in Ca 2÷-mobilizing cells by PKC has been proposed to be involved in homologous desensitization in DDT1 MF-2 cells [28] and vascular smooth muscle cells [11]. In this study, we have shown that P M A blocks BK receptor-mediated PI hydrolysis and Ca 2÷ mobilization in cultured canine TSMCs (Figs. 1 and 4). The concomitant loss o f hormonestimulated IPs accumulation and Ca 2÷ mobilization induced by short-term P M A treatment supports the causal relationship between these responses as suggested by previous studies [10, 11, 17, 29]. In canine TSMCs, IPs accumulation is a very early receptor-stimulated event. Our data indicate that IPs accumulation is a direct consequence of activation o f BK receptors in TSMCs, and that it is the inhibition of this response by P M A that prevents mobilization of Ca 2÷ from intracellular stores. Exposure o f the cells to P M A rapidly suppressed PKC in the cytosol but increased it transiently in the membranes, resulting in a decrease of the total PKC activity after 2 h. This result is consistent with the dual action o f P M A on PKC activity (Fig. 7). Phorbol esters are shown to activate PKC and increase the rate o f its degradation [31 ] with possible differences in sensitivities among isoforms [30]. P M A inhibited BK-induced IPs accumulation in cultured canine TSMCs in a concentration-dependent manner (Fig. 1). P M A did not affect the basal level of IP3, thus ruling out the

579

possibility that P M A caused its effect by depleting an agonist-sensitive pool of membrane PI. In addition, P M A did not change the resting level o f [Ca2+]i. The inhibitory effect o f P M A on BK-induced Ca 2+ mobilization appears to be directly related to the inhibition of IP3 formation. Because PKC activation is associated with several cellular responses, phorbol ester-mediated inhibition o f IP3 formation might occur at one or several different sites. In a number o f cell types, elevation of intracellular Ca 2+ by Ca 2÷mobilizing agonists known to act by receptormediated stimulation o f PI turnover has been shown to be inhibited by phorbol esters [10, 17, 19, 20, 29]. It has been suggested that protein phosphorylation mediated by interaction of phorbol esters with PKC may be the mechanism by which PMA modulates hormone-sensitive PI metabolism. According to some reports [33, 34], phorbol esters might attenuate a rise in IP3 through increasing its degradation by activation of a phosphomonoesterase specific for IP3. The activity of this cytosolic enzyme increases after phosphorylation by PKC which provides a mechanism for inhibiting the agonist-induced rise in IP3 accumulation in platelets. Phorbol esters that do not bind to or activate PKC, do not block histamine-stimulated IPs accumulation [19]. Our finding that PMA rapidly inhibits the BK-stimulated IPs accumulation and Ca 2+ mobilization is consistent with the view that P M A acts through activation o f PKC, since staurosporine, a potent PKC inhibitor, blocks the inhibitory effect of P M A (Fig. 6). Moreover, the inhibitory effect was reduced after incubating the cells for more than 4 h with PMA; this may be attributed to the biotransformation or inactivation o f P M A itself. Also, long-term treatment with P M A has been shown to enhance angiotensin II-stimulated PI hydrolysis in vascular smooth muscle cells [ 11 ]. This may be due to alter total incorporation and uptake of [3H]inositol into cells and inositol phospholipids. One site at which hormone-stimulated PI hydrolysis could be inhibited by P M A is located at the receptor level. It has been reported that

580

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phorbol esters induced phosphorylation of a~adrenergic receptors associated with antagonism o f PI b r e a k d o w n in DDT~ MF-2 cells and suggested that altered receptor binding m a y be a mechanism o f PKC-induced inhibitory effect [28]. Moreover, pretreatment of TSMCs with P M A for either 2 or 24 h did not change the Bm~ and KD o f BK receptors in canine TSMCs as c o m p a r e d with control (Table 2). It seems that BK receptors are not a site for the inhibitory effect of P M A on BK-induced responses. The ability of P M A to block the histamine-stimulated IPs accumulation also suggests that the target o f P M A is a more general c o m p o n e n t of the PI cycle than a specific m e m b r a n e receptor [20]. It is possible that P M A affects u n k n o w n transducers that couple receptor occupation to response. Several lines o f evidence suggest that a postreceptor site is the best unifying hypothesis for the location of the phorbol ester inhibitory effect. Phorbol esters blocked vasopressin-induced IP3 accumulation in A 10 cells without changing receptor binding and abolished guanine nucleotide shift, indicating that coupling o f the receptor to Gp was altered [35]. We also found that P M A blocks both BK- and A1F4-stimulated IPs accumulation in canine TSMCs [7]. Since P M A has no effect on the basal level of P I turnover, P K C can uncouple the G protein f r o m PLC. It has been shown that activation of P K C affects the G protein coupling process in neutrophils and platelets [36, 37] and inhibits the function of Giprotein in platelets [38]. In addition, activation o f P K C by phorbol esters has been shown to phosphorylate P I - P L C in rat basophilic leukemia cells, providing an additional mechanism for receptor-PLC uncoupling [39]. Regardless o f the precise mechanism, an implication of a postreceptor site o f phorbol ester inhibition is that other agonists which act through P I - P L C stimulation might be uncoupled f r o m cytosolic Ca 2+ mobilization. The inhibition of BK-stimulated increase of [Ca2+]i by P M A (Figs. 4 and 5) is in agreement with the inhibitory effect of P M A on agonistinduced IPs accumulation and Ca 2÷ mobilization in TSMCs [19, 20]. The fact that the same

experimental conditions block IP3 accumulation and C a 2+ mobilization suggest that the mechanism of this PKC-mediated inhibition is not confined to the IP3-sensitive Ca 2+ release site. This is consistent with the observation that the purified IP3 receptor f r o m brain is phosphorylated and not functionally modified by P K C [40]. In conclusion, we have demonstrated that short-term P M A treatment causes a negative feedback regulation on BK-induced IPs accumulation and Ca 2+ mobilization in canine TSMCs, while long-term P M A treatment is associated with a down-regulation of P K C and the loss of its inhibitory function. These results suggest that physiological activation o f P K C might serve as a modulator of cellular responses induced by IP3. The site of P M A inhibition appears to be at a postreceptor location and m a y be involved in P I - P L C itself, although several sites of P M A antagonism can not be excluded. To determine how D A G and other second messengers modulate agonist-induced cellular responses is important for clarifying the mechanisms underlying bronchial hyperreactivity and manipulating the contractile response of the airway in asthma. A ckno wledgements-- This work was supported by grant CMRP-340 from Chang Gung Medical Research Foundation and NSC84-2331-B182-040 to C. M. Y., and CMRP-403 and NSC84-2331-B182-091-M03 to S. F. L. from National Science Council, Taiwan, Republic of China. The authors are greatly indebted to Dr. Anthony Herp at Chang Gung College of Medicine and Technology for his critical reading of the manuscript and suggestions. Appreciation is also expressed to Dr. Delon Wu for his encouragement.

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