Regulation of prostacyclin production by [ Ca2+]i and protein kinase C in aortic smooth muscle cells A.-C. ERBRICH, Department
D. J. CHURCH,
M. B. VALLOTTON,
of Medicine, Division of Endocrinology,
Erbrich, A.-C., D. J. Church, M. B. Vallotton, and U. Lang. Regulation of prostacyclin production by [Ca”+]i and protein kinase C in aortic smooth muscle cells. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E800-E806,1992.-The respective roles of protein kinase C (PKC) and of cytosolic free Ca2+ concentration ([ Ca2+] i) in prostacyclin synthesis were investigated in aortic smooth muscle cells by using A23187 and phorbol 12-myristate 13-acetate (PMA) to bypass the hormonal receptor. Exposure of the cells to A23187 markedly increased prostacyclin production, which was’not affected by the PKC inhibitor staurosporine or by PKC depletion after prolonged incubation (48 h) of cells with PMA. The increase in [Ca”‘]i induced by A23187 did not affect membranous or cytosolic PKC activity in control and PMA-stimulated cells. Activation of PKC by PMA, a weak stimulant of prostacyclin production by itself, strongly potentiated A23 187-induced prostacyclin production, as well as that induced by the calcium-mobilizing hormone arginine vasopressin (AVP). The potentiating effect persisted for 30 min after the removal of PMA. However, this “memory” effect was not due to sustained levels of membranous PKC activity but probably to the prolonged influence of PKCinduced phosphorylation(s). Taken together, our results suggest that, although an increase in [Ca2+]i is sufficient for inducing prostacyclin production in rat aortic smooth muscle cells, activation of PKC is necessary for AVP-induced prostacyclin production in this same tissue. protein kinase C downregulation; calcium ionophore; prostaglandins CYTOSOLIC
FREE
CALCIUM
CONCENTRATION
( [Ca2+]i)
and protein kinase C (PKC) are known to be key factors in cellular signal transduction (3, 21). In aortic vascular smooth muscle cells, binding of arginine vasopressin (AVP) to its receptor (20) activates phospholipase C, which results in hydrolysis of phosphatidylinositol 4,5bisphosphate. This leads to an increase in D-myo-inositol 1,4,Strisphosphate (29) and 1,2-diacylglycerol, which triggers a transient rise of [ Ca2+]i (4, 20) and activates membranous PKC, respectively (5). These events appear to work synergistically, calcium mobilization being responsible for the initiation and PKC activation for the sustained phase (24) of the cellular response, namely contraction and prostacyclin production (1, 10, 18).
Phorbol esters such as 4@-phorbol 12-myristate 13acetate (PMA) can substitute for diacylglycerol and directly activate PKC, resulting in an increase in membranous PKC activity and in a corresponding decrease in cytosolic PKC activity (21). Activation of PKC by PMA does not affect basal [Ca2+]; in aortic smooth muscle cells but transiently inhibits AVP-induced D-myOinositol 1,4,Strisphosphate formation and the ensuing [Ca2+]i increase, an effect that contrasts with its potentiating effect on AVP-induced prostacyclin formation (6). However, prolonged exposure to PMA leads to the downregulation of PKC (17, 21, 26,30), probably due to increased proteolytic degradation of the membrane-asE800
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AND U. LANG
University Hospital, CH-1211 Geneva 4, Switzerland
sociated enzyme (30). In addition, the calcium ionophore A23187 stimulates prostacyclin production (13, 27) by a mechanism involving an influx of extracellular calcium into the cell, a response that is also strongly potentiated in the presence of PMA (18). In the present study, we investigated the respective roles of PKC and [Ca2+]; in prostacyclin production in aortic smooth muscle cells. To this purpose we evaluated the influence of PKC activation and PKC depletion on AVP- and A23187induced prostacyclin production and examined the effect of A23187 on membranous and cytosolic PKC activity. Results indicate that, although an increase in [Ca2+]; is sufficient to induce prostacyclin production per se, AVP-induced prostacyclin production requires the activation of PKC. MATERIALS
AND
METHODS
Materials. [y-“2P]ATP was obtained from Amersham International (Amersham, Bucks, UK). ATP, histone III-S, PMA, A23187 and 4-Br-A23187, guanosine 5’-O-(3-thiotriphosphate), 1,2-diolein, phosphatidylserine, dithiothreitol (DTT), y-globulin, leupeptin, collagenase, elastase, and Triton X-100 were purchased from Sigma (St. Louis, MO). Fura- acetoxymethyl ester (fura-2/AM) was obtained from Molecular Probes (Eugene, OR). DEAE-cellulose DE 52 was from Whatman (Maidstone, Kent, UK). AVP was a gift from Ferring (Malmo, Sweden). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and trypsin were from GIBCO (Bethesda, MD). The anti-6-ketoprostaglandin (PG) Flcv antiserum was kindly provided by Dr. M. J. Dunn (Division of Nephrology, Case Western Reserve University, Cleveland, OH). Preparation of rat aortic smooth muscle cells. Rat aortic smooth muscle cells were isolated as previously described by Capponi et al. (4). After dispersion, cells were plated in 90-mm plastic Petri dishes and cultured in DMEM containing 10% FCS. Cells were adherent after 24 h, and confluent monolayers were usually obtained after 7 days (cell density lo7 cells/Petri dish). Confluent cultures were passaged as previously described (4), and cells of the third to the fifth passage were used for all experiments described herein. Subcellular fractionation. After stimulation, cells were homogenized and cytosolic and solubilized particulate fractions were prepared as previously described (18). Unless specified otherwise, monolayers (4-6 Petri dishes) were washed with 20 mM tris(hydroxymethyl)aminomethane (Tris) . HCl buffer, pH 7.5, containing 2 mM EDTA, 10 mM ethylene glycol-bis(Paminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 mM DTT, 0.25 M sucrose, and 5 mM leupeptin (buffer A) and homogenized in 1.2 ml of the same buffer. Fifty microliters of the homogenate were collected for protein determination, and the rest was centrifuged at 100,000 g for 1 h at 4°C. The supernatant was used as the cytosolic fraction. The pellet was incubated for 30 min at 20°C with Triton X-100 (1% in buffer A), diluted to the original volume of the homogenate with buffer A (final Triton concentration 0.2%), and centrifuged at 100,000 g for 1 h. The supernatant obtained was used as the solubilized particulate fraction. Measurement of PKC actioity. Cytosolic and membranous cell
0 1992 the American
Physiological
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fractions were applied to DEAE-cellulose columns (2.5 x 0.9 cm), previously equilibrated in buffer B (in mM: 20 Tris HCl, pH 7.5,2 EDTA, 2 EGTA, and 10 DTT). Columnswere washed with equilibrating buffer, and PKC activity waseluted by means of a linear NaCl gradient (O-O.3 M) in buffer B. PKC eluates from DEAE-cellulose chromatography were assayedfor PKC as previously described(18). PKC activity wasdeterminedby measuring the transfer of 32P from [T-~~P]ATP to histone III-S. The final reaction mixture (240 ~1) contained 0.7-1.2 x lo6 counts/min [y- 32P]ATP 15 x 10m7 M ATP, 0.46 mM CaCl, 6.9 mM MgC12, and 3O’mg histone III-S. Each fraction was assayedin the presenceand absenceof phosphatidylserine (80 pg) and diolein (4 pg), added as a lipid suspensionin 100 mM Tris HCl, pH 7.5. The reaction was carried out at 30°C for 10 min and wasstoppedby adding 2 ml of 12% trichloroacetic acid (TCA) in the presenceof 600 pg y-globulin added as carrier. After centrifugation the pellet was dissolved in 0.5 ml 1 N NaOH and precipitated again with 12% TCA. After a second centrifugation the protein precipitate was dissolved in 1 N NaOH and 32P incorporation was measuredby scintillation counting in Ultima Gold (Packard). Measurement of [Ca2+ji. [Ca2+]i was measuredin confluent monolayerswith the fluorescent Ca2+probe fura-2/AM, adapting the method describedelsewhere(4). Cells were grown on glasscover slips in DMEM with 2% FCS. When confluencewas attained, the cells were washed twice in Krebs-Ringer buffer (KRB). The monolayerswere then covered with 400 ~1of KRB containing 2 PM fura- and 0.5% bovine serumalbumin (BSA) and wereincubated for 30 min at 37°C. At the end of the loading period, the slideswere washedtwice in KRB without albumin and inserted into glasscuvettes (1 x 1 cm) then placed in the thermostated holder of an LS-3 Perkin-Elmer fluorescence spectrophotometer. Continuous stirring was achieved with a magnetic stirrer. Fluorescenceof fura-2-loaded monolayerswas measuredusing excitation wavelength 339 nm and emission wavelength 505 nm. Calibration of the signal was performed with ionomycin and MnC12in the presenceof an excessof Ca2+, as reported elsewhere(4). Functional tests. Aortic smooth muscle cells seeded into 90-mm Petri or 6-well tissue culture plates were washedand incubated for 30 min at 37°C with 10 ml or 1 ml, respectively, with KRB buffer containing 0.2% BSA and 0.2% glucose.The supernatant was then replaced with fresh buffer, and the cells were incubated at 37°C with 95% air-5% CO, in the presenceof various stimuli. At the end of the incubation period, the prostacyclin content of the media was determined by a specific radioimmunoassayof its stable metabolite 6-keto-PGF,,,, as previously described (18). The cross-reactivity of the anti-6keto-PGF,,, rabbit antiserum was 14% with PGF1,,, 2% with PGE,, 1.7% with PGF,,,, and ~1% with the other major arachidonic acid metabolites.The intra- and interassaycoefficients of variation of the assay were 4 and 8%, respectively, and the sensitivity of the assaywas 3 pg. Statistical analysis. Student-Fisher unpaired bilateral t tests, analysis of variance using the Scheffe F test criterion for unbalanced groups, and principal component (factor) analysis by the method of least-squaresfit were usedwhere applicable. The data represent meanst SE of at least three experiments performed in duplicate or triplicate determinations.
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RESULTS
Influence of PKC activation and [Ca2+]i elevation on prostacyclin production in aortic smooth muscle cells. Fig-
ure 1 shows the effect of maximal concentrations of AVP, A23187, and PMA on PKC activity and on prostacyclin production. Incubation with AVP (10m7 M) for 30 min enhanced prostacyclin production from 0.2 t 0.02 to 4.1
membranes 0
Basal
m
A23187
m
PMA A23187
0
non-treated
+ PMA
AVP
48h PMA
Fig. 1. Effect of A23187 and arginine vasopressin (AVP) on protein kinase C (PKC) activity and prostacyclin production in intact and PKC-depleted aortic smooth muscle cells. Aortic smooth muscle cells were pretreated with 100 nM PMA for 48 h and then incubated for 30 min with A23187 (5 X lOA M) or AVP (10s7 M). Prostacyclin production (A) and membranous and cytosolic PKC activity (B and C) were determined as described in MATERIALS AND METHODS. PKC activity is expressed as percentage of control values. Values are means t SE of 4 separate experiments. PMA, phorbol 12-myristate 13-acetate.
+ 0.3 ng/mg protein (P < 0.001, n = 3). The ionophore A23187 (5 X 10B6 M) increased prostacyclin formation to 3.5 t 0.2 ng/mg protein (P < 0.001, n = 3), whereas the PKC activator PMA (lo- 7 M) stimulated prostacyclin synthesis only to 1.0 t 0.1 ng/mg protein (P < 0.001, n = 3). However, PMA potentiated the A23187-induced prostacyclin formation by 240 t 14% (P c 0.001, n = 3, vs. A23187 alone). Exposure to PMA (10m7 M) for 30 min enhanced basal membranous PKC activity by 209 t 26% (P < 0.001, n = 4), whereas cytosolic PKC activity was decreased by 73 t 5% (P < 0.001, n = 4). AVP ( 10S7 M) increased membranous PKC activity by 52 t 12% (P < 0.01, n = 3) but had no significant effect on cytosolic PKC activity. In contrast, A23187 (5 x 10S6 M) did not modify membranous or cytosolic PKC activity in control and PMA-stimulated cells (Fig. 1). To determine whether A23187 could induce membranous PKC association, which dissociates when the 10 mM EGTA present in our homogenization buffer is added (see MATERIALS AND METHODS), we further measured PKC activity in cytosolic and membranous fractions prepared in the absence of EGTA. When cells were homogenized in the absence of EGTA, A23187 (5 x 10B6 M) appeared to induce a small but statistically insignificant increase in membranous PKC activity (Table 1).
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Table 1. Effect of EGTA on cytosolic and membranous PKC association in homogenates of A23187and PMA-stimulated cells Protein Treatment
Control A23187 PMA
+lO
mM
Kinase
C Activity,
cpm/mg
EGTA
protein
Without
EGTA
Cytosol
Membranes
Cytosol
Membranes
3,673+375 3,530&414 561k84
378t48 411t55 1,454+156
2,034+427 2,641&461 488t55
714t64 926&103 1,416+111
Values are means t SE of 4 separate experiments. Aortic smooth muscle cells were incubated for 30 min with phorbol 12-myristate 13acetate (PMA, 1O-7 M) or A23187 (5 x lo-(’ M) and membranous and cytosolic cell fractions were prepared in presence or absence of 10 mM EGTA. Protein kinase C (PKC) activity was determined as described in MATERIALS AND METHODS. PKC activity is expressed in counts per into histone III-S per 10 min per minute (cpm) of :32P incorporated milligram protein homogenate.
Long-term exposure to phorbol ester is known to desensitize PKC activity (26). In aortic smooth muscle cells, prolonged preincubation for 48 h with PMA (10M7 M) completely suppressed PKC activity as follows: it was no longer possible to detect any PKC activity, neither in control nor in AVP- or PMA-stimulated cells (Fig. 1). In these PKC-depleted cells, PMA did not stimulate prostacyclin production, whereas AVP-induced prostacyclin production was inhibited by 74 t 2% (P < 0.01, n = 3). In contrast, although A23187-induced prostacyclin production was not affected by PKC depletion, it was no longer potentiated by PMA. These observations suggest that, although an elevation of [Ca2+]i is a sufficient stimulus for prostacyclin production in these cells, PKC plays an important modulatory role during hormonal stimulation. This conclusion was further substantiated by the finding that staurosporine (5 x 10S7 M), which inhibited membranous and cytosolic PKC activity in both control- (85 and 64% inhibition, respectively) and PMA-stimulated cells (93 and 58% inhibition, respectively), did not inhibit A23187-induced prostacyclin production yet abolished the PMA-induced response in the presence and absence of A23187 (P < 0.005, n = 3, Fig. 2). Identical results were 0 I
q
C
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obtained with the highly selective PKC inhibitor CGP41251 (10e6 M, see Ref. 19), a compound that had no significant effect on the A23 187-stimulated response yet also inhibited the effect of PMA in the presence and absence of A23187 (P < 0.005, n = 4). To assess the effect of A23187 on cellular [Ca2+]i, we measured [Ca2+]i with the fluorescent probe fura-2/AM using the nonfluorescent derivative of A23187, 4-BrA23187. The addition of 4-Br-A23187 (5 x low6 M) resulted in an increase in [Ca2+]; from a resting value of 141 t 13 to 471 t 80 nM (n = 5) and lasting up to 3 min while AVP ( 10e7 M) induced a biphasic rise in [Ca2+]i, the transient [Ca”+]i spike recorded at 455 t 52 nM (n = 5) followed by a lower sustained phase as has previously been reported (see Ref. 6). Overall, 4-Br-A23187 was found to be a less potent stimulus for prostacyclin production than A23187. Maximal concentration of 5 X lo-” M 4-Br-A23 187 stimulated prostacyclin production from 0.12 t 0.02 to 0.45 t 0.06 ng/mg protein (P < 0.01, n = 3)) whereas A23 187 increased prostacyclin production to 1.30 t 0.03 ng/mg protein (P < 0.001, n = 3). Effect of short- and long-term PMA treatment on A23187- and A VP-induced prostacyclin production in rat aortic smooth muscle cells. Pretreating cells with 10S7 M
PMA resulted in a time-dependent potentiation of both the A23187- and the AVP-induced prostacyclin production, with maximal augmentations of 454 t 15 and 487 t 31% at 1 h, respectively (Fig. 3). Thus PKC activation by PMA appears to modulate AVP- and A23187-stimulated prostacyclin formation. Longer periods of preincubation with PMA resulted in a decrease of the PMA-induced potentiation. After 48 h of exposure to PMA, the A23187induced response was similar to that observed in nonpreincubated cells, whereas AVP-stimulated prostacyclin production was inhibited by 56 t 9% (Fig. 3). Prolonged effect of PKC activation on AVP- and A23187-induced prostacyclin production. Because PKC
activation is known to be responsible for the sustained phase of the biological response in many systems, we determined whether the PMA-induced activation of PKC and its potentiating effect on A23187- or AVP-induced
wlthout Inhibitor + staurosporine + CGP41251
A23
PMA
A23+PMA
Fig. 2. Effect of staurosporine and CGP-41251 on PMAand A23187induced 6-keto-PGF,,, production in aortic smooth muscle cells. Cells were stimulated for 30 min with PMA (10m7 M), A23187 (5 X 10ec’ M), or PMA and A23187 in absence or presence of staurosporine (5 X 10Y7 M) or CGP-41251 (lo-” M). 6-keto-PGF,(, production was determined as described in MATERIALS AND METHODS. Values are means * SE of 4 independent experiments.
0
1
2 Preincubation
3 24 with PMA (hours)
48
Fig. 3. Effect of PMA on A23187and AVP-induced prostacyclin production. Aortic smooth muscle cells were preincubated with 100 nM PMA for indicated times before being stimulated with A23187 (5 X lo-” M) or AVP (lOhY M) for 30 min. 6-Keto-PGF,,, production was determined as described in MATERIALS AND METHODS. Values are means + SE of 3-4 separate experiments.
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prostacyclin formation persisted after the removal of PMA. In cells pretreated for 30 min with 10m7 M PMA, thus having PKC maximally activated (18), and then washed free of PMA, the response to the immediate addition of A23187 (Fig. 4B) or AVP (Fig. 4C) was potentiated by 548 t 109% (P < 0.01, n = 3) and 141 t 19% (P < 0.01, n = 3), respectively. This potentiating effect was still present 30 min after removal of PMA, before stimulation with A23187 or AVP (Fig. 4, B and C). After a l-h interval without PMA, the potentiating effect of PMA decreased to 256 t 50% of A23187-stimulated prostacyclin production in nonpretreated cells (P < 0.05 vs. A23187 alone, n = 3, Fig. 4B). Interestingly, 10m7 M PMA potentiated A23 187-induced prostaglandin release in cells previously pretreated with phorbol ester and allowed to rest for 60 min (Fig. 4, E vs. B), a response similar to that obtained in cells having been stimulated with A23187
IN AORTIC
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immediately after 30 min of preincubation with PMA (Fig. 4B). In contrast to what was observed in A23187-stimulated cells, the potentiating effect of PMA pretreatment on the AVP-stimulated prostacyclin response was further increased after 60 min of incubation in the absence of PMA, reaching 363 t 33% of the AVP-induced response in nonpretreated cells (Fig. 4C). The presence of 10e7 M PMA during AVP stimulation did not further augment the AVP-induced response in these cells (Fig. 4F). When cells were preincubated with PMA for time periods of 5 or 10 min, which induce submaximal activation of PKC (6, 13), A23187-stimulated prostacyclin production was still augmented by 483 t 19 and 483 t 88%, respectively, compared with a potentiation of 1,405 t 41% in cells pretreated for 30 min. (Fig. 5). These weaker potentiating effects could still be observed after 30 min of incubation without PMA, before stimulation with A23187 (Fig. 5). In contrast, preincubation of cells with A23187 (5 x lo- ci M) for 30 min had no potentiating effect on PMA-induced prostacyclin production, even when stimulation occurred immediately after the end of A23187 pretreatment (data not shown). Relation between PKC activation and prostacyclin production. To investigate whether the persistence of the
effect of PMA on prostacyclin production 15-30 min after removal of PMA was due to persistent PKC activation, we compared membranous PKC activity with A23187-induced prostacyclin formation. Figure 6 shows that there was no strong correlation between membranous PKC activity and the potentiating effect of PMA pretreatment on A23187-induced prostacyclin formation. ‘, ,’
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Fig. 4. Effect of various time intervals between initial PMA stimulation and addition of A23187 or AVP on subsequent prostacyclin response. Aortic smooth muscle cells were preincubated with PMA (lo-’ M) for indicated times. Medium was then removed and replaced by fresh Krebs-Ringer buffer (MATERIALS AND METHODS). Cells were either incubated immediately with A23187 (5 x lo-(’ M) or AVP (10Y7 M) for 30 min in presence (hatched bars) or absence (open bars) of PMA ( 10Y7 M) or were allowed to recuperate for indicated times before stimulation. Values are means t SE of 3-4 separate experiments. A: control. B: A23187 (5 x lo-(; M). C: AVP (1O-7 M). D: PMA (1OW M). E: A23187 (5 x lo-(; M) + PMA (lo+ M). F: AVP (1O-Y M) + PMA (1O-7 M).
6
PMA 30 min
4 2 0 b
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Fig. 5. Effect of changes in duration of quent prostacyclin response induced by smooth muscle cells were pretreated with 30 min. Cells were then either stimulated 3O-min recuperation period in absence means k SE of 4 separate experiments.
30 min interval
PMA pretreatment on subseA23187 30 min later. Aortic PMA ( 10m7 M) for 0,5, 10, or immediately (left) or after a of PMA (right). Values are
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to be inversely correlated with A23 187-induced prostacyclin production (r = -0.95, P < 0.005, n = 5), suggesting that the level of cytosolic PKC activity is a robust indicator for the degree of activation of the phosphorylation cascade leading to the prostacyclin response. DISCUSSION
3
x -5 -
IN AORTIC
*
’
100 80 60
~_ Untreated
PMA 30min
.PMA 30mln
PMA 30mln
P;A 30mln
P;A 60mln
Fig. 6. Relationship between PKC activity and A23187-induced prostacyclin production. Aortic smooth muscle cells were pretreated with PMA (10v7 M) for 30,60, or 90 min either continuously, or, after 30 min of PMA-pretreatment, cells were incubated for another 30 or 60 min without PMA. Cells were then either stimulated with A23187 (5 X lo-” M) for 30 min and prostacyclin production was measured (A), or membranous and cytosolic PKC activities were determined (B and C, respectively). 6-Keto-PGF,,r and PKC activity were measured as described in MATERIALS AND METHODS. PKC activity is expressed as percentage of control values. Values are means k SE of 4 separate experiments.
Membranous PKC activity was maximally increased, to 450 t 43% of control values after 30 min of PMA treatment. Longer exposures to PMA for 60 and 90 min decreased membranous PKC activity to 271 t 19 and 176 t 13%, respectively, of control values. In contrast, A23187induced prostacyclin production was similar in cells pretreated for 30 or 60 min with PMA, and a longer preincubation period of 90 min augmented the PMA potentiating effect by 26 t 5% (Fig. 6). When cells that had been exposed for 30 min to PMA were incubated for 30 and 60 min without PMA, membranous PKC activity was still increased to 409 t 38 and 282 t 54%, respectively (P < 0.001 and P < 0.01 vs. control; n = 4 and 6, respectively). However, the potentiating effect of PMA pretreatment on prostacyclin production was significantly decreased (P < 0.05 and P < 0.01; n = 3 and 4, respectively) when compared with that of cells immediately stimulated after PMA pretreatment (Fig. 6). Thus membranous PKC activity cannot be correlated with A23187-stimulated prostacyclin production. This suggests that the prolonged effect of PMA on A23187-induced prostacyclin production is not due to high levels of membranous PKC activity but to an event following PKC activation. In contrast, cytosolic PKC activity was found
Activation of PKC and intracellular calcium mobilization are known to be essential intracellular events for various physiological processes (14, 16, 21). To investigate the respective roles of [Ca2+]i and PKC in aortic smooth muscle cells during AVP stimulation, we first determined the influence of A23187-induced [Ca2+]i elevation on PKC activation. In the presence of 1.2 mM extracellular Ca2+, the nonfluorescent A23 187 analogue 4-Br-A23187 was shown to induce a similar but more sustained increase in [Ca2+]i compared with that observed with AVP. We did not observe a significant effect of A23187 on membranous and cytosolic PKC activity in control or in PMA-stimulated aortic smooth muscle cells. Studies on the influence of A23187 on PKC activity are controversial. A23187 was found to increase membranous PKC activity in leukocytes (7) and in pinealocytes (14). It was also shown to enhance the binding of phorbol12,13-dibutyrate ([3H]PDB) to phagocyte (8), platelet (25), and leukemia cell (30) membranes, a result that is considered to correspond to an increase in membranous PKC activity. However, Saitoh et al. (25), studying the influence of epinephrine and A23187 on both [3H]PDB binding to membranous PKC and phosphorylation of a 47-kDa protein (a PKC-specific substrate) in platelets, have observed that epinephrine increased both [3H]PDB binding to membranous PKC and 47-kDa protein phosphorylation. In contrast, although A23187 increased [“H]PDB binding to membranous platelet PKC, it did not affect phosphorylation of the 47-kDa protein. Thus an increase in the [“H]PDB binding to membranous PKC does not always appear to reflect fully activated PKC. Various types of PKC association with cellular membranes have been suggested as follows: one that is entirely Ca2’ dependent and dissociable by the addition of EGTA, one that is induced by an increase in diacylglycerol and [Ca2*]i, and one that is induced by phorbol esters in the absence of an increase in [ Ca2+] i (11, 14). An elevation of [ Ca2+] i could induce PKC redistribution from the cytosol to the-membrane but would be insufficient to fully activate the membrane-bound PKC. The presence of diacylglycerol or phorbol esters, such as PMA, appears to be necessary to promote PKC penetration in membranous phospholipids to induce the conformational change resulting in a fully active lipid-protein complex (9). In addition, Christiansen et al. (7) have reported that, in platelets, the increase in [Ca2’]i has to be very high to induce the subcellular redistribution of PKC. Ionomycin, which increases [Ca2*]; to 2 mM in these cells, also induces the translocation of PKC to the membranous fraction, whereas N-formylmethionylleucylphenylalanine, which only increases [Ca2*]; to 500 nM, has no effect on membranous PKC activity (7). Thus the A23187-stimulated rise in [Ca2+]i (from 14 t 13 to 471 t 80 nM) observed in aortic smooth muscle cells could well be insufficient for
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OF PGI,
PRODUCTION
PKC activation in these cells. Nevertheless, A23187 was found to be a potent stimulus for prostacyclin production in aortic smooth muscle cells. Its stimulating action was found to be independent of PKC activation, since it was not affected by the PKC inhibitor staurosporine or by prolonged treatment (48 h) with PMA resulting in PKC depletion. These observations suggest that [ Ca2+]i itself plays an important role in prostacyclin production in aortic smooth muscle cells, probably by activating a calcium-sensitive phospholipase. In contrast, the PMA-induced potentiation of A23187induced prostacyclin release was abolished by both staurosporine and the highly selective PKC inhibitor CGP41251; it was also no longer present in PKC-depleted cells. In these cells, AVP-stimulated prostacyclin production was furthermore decreased by 70% when compared with nonpretreated cells. These results indicate that PKC plays a crucial modulatory role during hormonal stimulation of prostacyclin production in aortic smooth muscle cells. They are confirmed by a study of Pirotton et al. (23) who described a decrease of ATP- but not of A23187induced [ 3H] choline release in bovine aortic endothelial cells treated for 24 h with PMA. Kanterman et al. (15) observed that downregulation of PKC with long-term (24 h) PMA treatment in dopamine receptor-transfected ovary cells not only abolished the dopamine-induced stimulatory effect but also partially decreased the A23187-stimulated arachidonic acid release (15). The potentiating influence of PKC activation on A23 187-induced prostacyclin production persisted for -30 min after the removal of PMA, whereas in AVPstimulated cells, it was still present 1 h after the removal of PMA. This difference can be explained by the fact that A23187 induces only an elevation of [Ca2+]i without affecting PKC activity, whereas AVP acts through phosphatidylinositol4,5-bisphosphate hydrolysis, thus elevating both [ Ca2+]i and membranous PKC activity. Previous studies (28) have shown that angiotensin II (ANG II)induced PGI, production is potentiated by a previous exposure to AVP. This potentiating effect persists for 90 min after the removal of AVP, suggesting that a postreceptor event such as PKC activation is implicated. Barrett et al. (2) have shown that previous activation of adrenal glomerulosa cells with ANG II potentiates a second ANG II-induced aldosterone secretion even after a time interval of 35-60 min in the absence of ANG II. They noticed that this potentiating effect persisted only when the first stimulation with ANG II was long enough (20 min) to maximally stimulate PKC activity. In contrast, the potentiating effect of PMA on aortic smooth muscle cells persists also when the cells have been incubated for time periods (5-10 min) that do not allow for full PKC activation. The use of different stimuli could explain the discrepancy between our results and those of Barrett et al. (2). In summary, our determinations of PKC activity and prostacyclin production in aortic smooth muscle cells suggest that the “memory” effect of PMA-induced PKC activation is not due to sustained levels of membranous PKC activity but is probably linked to the phosphorylation of protein(s) after PKC activation.
IN AORTIC
SMOOTH
MUSCLE
E805
CELLS
We thank C. Gerber-Wicht, M. Klein, and M. Rey for excellent technical assistance and Dr A. M. Capponi for helpful discussion. This study was supported by Grant 31.21727.89 from the Swiss National Science Foundation. D. J. Church was further supported by a postgraduate scholarship from the Glaxo Institute of Molecular Biology, Geneva, Switzerland. Address for reprint requests: U. Lang, Div. of Endocrinology, Geneva Univ. Hospital, 24, Rue Micheli-du-Crest, CH-1211 Geneva 4, Switzerland. Received
29 October
1991; accepted
in final
form
22 May
1992.
REFERENCES 1. Altura, B. M., and B. T. Altura. Actions of vasopressin, ocytocin and synthetic analogs on vascular smooth muscle. Federation Proc. 43: 80-86, 1984. 2. Barrett, P. Q., I. Kojima, K. Kojima, K. Zawadich, C. M. Isales, and H. Rasmussen. Short term memory in the calcium messenger system. Biochem. J. 238: 905912, 1986. 3. Berridge, M. B. Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220: 345-360, 1987. 4. Capponi, A. M., P. D. Lew, and M. B. Vallotton. Cytosolic free calcium levels in monolayers of cultured rat aortic smooth muscle cells. J. BioZ. Chem. 260: 7836-7842, 1985. 5. Chardonnens, D., U. Lang, A. M. Capponi, and M. B. Vallotton. Comparison of the effects of angiotensin II and vasopressin on cytosolic free calcium concentration, protein kinase C activity and prostacyclin production in cultured rat aortic and mesenteric smooth muscle cells. J. Cardiovusc. Pharmacol. 14, Suppl. 6: S39-S44, 1989. 6. Chardonnens, D., U. Lang, M. F. Rossier, A. M. Capponi, and M. B. Vallotton. Inhibitory and stimulatory effects of phorbol ester on vasopressin induced cellular response in cultured rat aortic smooth muscle cells. J. Biol. Chem. 265: 10451-10457, 1990. 7. Christiansen, N. O., C. S. Larsen, and V. Esmann. A study on the role of protein kinase C and intracellular calcium in the activation of superoxide generation. Biochim. Biophys. Actu 971: 317-324, 1988. 8. Dougerty, R. W., and J. E. Niedel. Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes. J. BioZ. Chem. 261: 4097-4100, 1986. 9. Epand, R. M., and D. S. Lester. The role of membrane biophysical properties in the regulation of protein kinase C activity. Trends Phurmucol. Sci. 11: 317-320, 1990. 10. Forder, J., A. Scriabine, and H. Rasmussen. Plasma membrane calcium flux, protein kinase C activation and smooth muscle contraction. J. Phurmucol. Exp. Ther. 235: 267-273, 1983. 11. Gopalakrishna, R., S. H. Barsky, T. P. Thomas, and W. B. Anderson. Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca”+-induced and phorbol ester-stabilized membrane binding of protein kinase C. J. BioZ. Chem. 261: 16438-16445, 1986. 12. Grynkiewicz, G., M. Peonie, and M. Y. Tsien. A new generation of Ca”+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450, 1985. 13. Hassid, A., and J. P. Oudinet. Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells. Prostuglundins 32: 457-478, 1986. 14. Ho, A. K., T. P. Thomas, C. L. Chik, W. B. Anderson, and D. C. Klein. Protein kinase C: subcellular redistribution by increased Ca2+ influx. J. Biol. Chem. 263: 9292-9297, 1988. 15. Kanterman, R. Y., L. C. Mahan, E. M. Briley, F. J. Monsma, D. R. Sibley, J. Axelrod, and C. C. Felder. Transfected D2 dopamine receptors mediate the potentiation of arachidonic acid release in Chinese hamster ovary cells. Mol. Phurmucol. 39: 364-369, 1991. 16. Kishimoto, U., Y. Takai, T. Mori, U. Kikkawa, and Y. Nishizuka. Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol: its possible relation to phosphatidylinositol turnover. J. Biol. Chem. 255: 2273-2276, 1980. 17. Lang, U., D. Fabbro, D. Chardonnens, and M. B. Vallotton. Two types of protein kinase C modulate angiotensin IIinduced prostacyclin production in aortic smooth muscle cells
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E806 (Abstract).
REGULATION
OF PGI,
PRODUCTION
IN AORTIC
Proc. Annu. Meet. Endow. Sot. 11th Washington DC
1991, p. 86. 18. Lang, U., and M. B. Vallotton. Effects of angiotensin II and of phorbol ester on protein kinase C activity and on prostacyclin production in cultured rat aortic smooth muscle cells. Biochem. J. 259: 477-484, 1989. 19. Meyer, T., U. Regenass, D. Fabbro, E. Alteri, J. Rosel, M. Muller, G. Caravatti, and A. Matter. A derivative of staurosporine (CGP 41251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-tumor activity. Int. J. Cancer 43: 851-856, 1989. 20. Nabika, T., P. A. Velletri, W. Lovenberg, and M. A., Beaven. Increase in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arglvasopressin in vascular smooth muscle cells. J. Biol. Chem. 260: 4661-4670, 1985. 21. Nishizuka, Y., A. P. Sloan, and J. R. Prize. Studies and perspectives of the protein kinase C family for cellular regulation. Cancer 63: 1892-1903, 1989. 22. Penit, J., M. Faure, and S. Jard. Vasopressin and angiotensin II receptors in rat aortic smooth muscle cells in culture. Am. J. Physiol. 244 (Endocrinol. Metab. 7): E72-E82, 1983. 23. Pirotton, S., B. Robaye, C. Lagneau, and J.-M. Boeynaems. Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells. J. CeLI. Physiol. 142: 449-457, 1990. 24. Rasmussen, H., Y. Takuwa, and S. Park. Protein kinase C in the regulation of smooth muscle contraction. FASEB J. 1: 177185, 1987. 25. Saitoh, M., E. W. Salzman, M. Smith, and J. A. Ware. Activation of protein kinase C in platelets by epinephrine and
26.
27.
28.
29.
30.
31.
SMOOTH
MUSCLE
CELLS
A23187: correlation with fibrinogen binding. Blood 74: 2001-2006, 1989. Stassen, F. L., D. B. Schmidt, M. Papadopoulos, and H. M. Sarau. Prolonged incubation with phorbol esters enhanced vasopressin-induced calcium mobilization and phosphatidylinositol hydrolysis of vascular smooth muscle cells. J. Biol. Chem. 264: 4916-4923, 1989. Taylor, G. W., C. G. Chappel, C. E. Frazer, and J. M. Ritter. Prostacyclin synthesis by vascular tissue. Effect of extracellular Ca ‘2+. Biochem. J. 239: 237-239, 1986. Vallotton, M. B., C. Gerber-Wicht, W. Dolci, and R. P. Wuthrich. Interaction of vasopressin and angiotensin II in stimulation of prostacyclin synthesis in vascular smooth muscle cells. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E617-E624, 1989. Vittet, D., P. Berta, M.-N. Mathieu, A. Rondot, P. Trovo, B. Cantau, and C. Chevillard. Vlcr vasopressin-induced accumulation of inositol trisphosphate in cultured rat aortic myocytes: modulation by protein kinase C. Biochem. Biophys. Res. Commun. 140: 1093- 1100, 1980. Young, S., P. J. Parker, A. Ullrich, and S. Stabel. Downregulation of protein kinase C is due to increased rate of degradation. Biochem. J. 244: 775-779, 1987. Zor, U., E. Her, T. Harell, G. Fischer, Z. Naor, P. Braquet, E. Ferber, and N. Reiss. Arachidonic acid release by basophilic leukemia cells and macrophages stimulated by Ca*+ ionophores, antigen and diacylglycerol: essential role for protein kinase C and prevention by glucocorticoids. Biochim. Biophys. Acta 1091: 385392, 1991.
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D. J. CHURCH,
M. B. VALLOTTON,
of Medicine, Division of Endocrinology,
Erbrich, A.-C., D. J. Church, M. B. Vallotton, and U. Lang. Regulation of prostacyclin production by [Ca”+]i and protein kinase C in aortic smooth muscle cells. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E800-E806,1992.-The respective roles of protein kinase C (PKC) and of cytosolic free Ca2+ concentration ([ Ca2+] i) in prostacyclin synthesis were investigated in aortic smooth muscle cells by using A23187 and phorbol 12-myristate 13-acetate (PMA) to bypass the hormonal receptor. Exposure of the cells to A23187 markedly increased prostacyclin production, which was’not affected by the PKC inhibitor staurosporine or by PKC depletion after prolonged incubation (48 h) of cells with PMA. The increase in [Ca”‘]i induced by A23187 did not affect membranous or cytosolic PKC activity in control and PMA-stimulated cells. Activation of PKC by PMA, a weak stimulant of prostacyclin production by itself, strongly potentiated A23 187-induced prostacyclin production, as well as that induced by the calcium-mobilizing hormone arginine vasopressin (AVP). The potentiating effect persisted for 30 min after the removal of PMA. However, this “memory” effect was not due to sustained levels of membranous PKC activity but probably to the prolonged influence of PKCinduced phosphorylation(s). Taken together, our results suggest that, although an increase in [Ca2+]i is sufficient for inducing prostacyclin production in rat aortic smooth muscle cells, activation of PKC is necessary for AVP-induced prostacyclin production in this same tissue. protein kinase C downregulation; calcium ionophore; prostaglandins CYTOSOLIC
FREE
CALCIUM
CONCENTRATION
( [Ca2+]i)
and protein kinase C (PKC) are known to be key factors in cellular signal transduction (3, 21). In aortic vascular smooth muscle cells, binding of arginine vasopressin (AVP) to its receptor (20) activates phospholipase C, which results in hydrolysis of phosphatidylinositol 4,5bisphosphate. This leads to an increase in D-myo-inositol 1,4,Strisphosphate (29) and 1,2-diacylglycerol, which triggers a transient rise of [ Ca2+]i (4, 20) and activates membranous PKC, respectively (5). These events appear to work synergistically, calcium mobilization being responsible for the initiation and PKC activation for the sustained phase (24) of the cellular response, namely contraction and prostacyclin production (1, 10, 18).
Phorbol esters such as 4@-phorbol 12-myristate 13acetate (PMA) can substitute for diacylglycerol and directly activate PKC, resulting in an increase in membranous PKC activity and in a corresponding decrease in cytosolic PKC activity (21). Activation of PKC by PMA does not affect basal [Ca2+]; in aortic smooth muscle cells but transiently inhibits AVP-induced D-myOinositol 1,4,Strisphosphate formation and the ensuing [Ca2+]i increase, an effect that contrasts with its potentiating effect on AVP-induced prostacyclin formation (6). However, prolonged exposure to PMA leads to the downregulation of PKC (17, 21, 26,30), probably due to increased proteolytic degradation of the membrane-asE800
0193-1849/92
$2.00
Copyright
AND U. LANG
University Hospital, CH-1211 Geneva 4, Switzerland
sociated enzyme (30). In addition, the calcium ionophore A23187 stimulates prostacyclin production (13, 27) by a mechanism involving an influx of extracellular calcium into the cell, a response that is also strongly potentiated in the presence of PMA (18). In the present study, we investigated the respective roles of PKC and [Ca2+]; in prostacyclin production in aortic smooth muscle cells. To this purpose we evaluated the influence of PKC activation and PKC depletion on AVP- and A23187induced prostacyclin production and examined the effect of A23187 on membranous and cytosolic PKC activity. Results indicate that, although an increase in [Ca2+]; is sufficient to induce prostacyclin production per se, AVP-induced prostacyclin production requires the activation of PKC. MATERIALS
AND
METHODS
Materials. [y-“2P]ATP was obtained from Amersham International (Amersham, Bucks, UK). ATP, histone III-S, PMA, A23187 and 4-Br-A23187, guanosine 5’-O-(3-thiotriphosphate), 1,2-diolein, phosphatidylserine, dithiothreitol (DTT), y-globulin, leupeptin, collagenase, elastase, and Triton X-100 were purchased from Sigma (St. Louis, MO). Fura- acetoxymethyl ester (fura-2/AM) was obtained from Molecular Probes (Eugene, OR). DEAE-cellulose DE 52 was from Whatman (Maidstone, Kent, UK). AVP was a gift from Ferring (Malmo, Sweden). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and trypsin were from GIBCO (Bethesda, MD). The anti-6-ketoprostaglandin (PG) Flcv antiserum was kindly provided by Dr. M. J. Dunn (Division of Nephrology, Case Western Reserve University, Cleveland, OH). Preparation of rat aortic smooth muscle cells. Rat aortic smooth muscle cells were isolated as previously described by Capponi et al. (4). After dispersion, cells were plated in 90-mm plastic Petri dishes and cultured in DMEM containing 10% FCS. Cells were adherent after 24 h, and confluent monolayers were usually obtained after 7 days (cell density lo7 cells/Petri dish). Confluent cultures were passaged as previously described (4), and cells of the third to the fifth passage were used for all experiments described herein. Subcellular fractionation. After stimulation, cells were homogenized and cytosolic and solubilized particulate fractions were prepared as previously described (18). Unless specified otherwise, monolayers (4-6 Petri dishes) were washed with 20 mM tris(hydroxymethyl)aminomethane (Tris) . HCl buffer, pH 7.5, containing 2 mM EDTA, 10 mM ethylene glycol-bis(Paminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 mM DTT, 0.25 M sucrose, and 5 mM leupeptin (buffer A) and homogenized in 1.2 ml of the same buffer. Fifty microliters of the homogenate were collected for protein determination, and the rest was centrifuged at 100,000 g for 1 h at 4°C. The supernatant was used as the cytosolic fraction. The pellet was incubated for 30 min at 20°C with Triton X-100 (1% in buffer A), diluted to the original volume of the homogenate with buffer A (final Triton concentration 0.2%), and centrifuged at 100,000 g for 1 h. The supernatant obtained was used as the solubilized particulate fraction. Measurement of PKC actioity. Cytosolic and membranous cell
0 1992 the American
Physiological
Society
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REGULATION
OF PG12 PRODUCTION
fractions were applied to DEAE-cellulose columns (2.5 x 0.9 cm), previously equilibrated in buffer B (in mM: 20 Tris HCl, pH 7.5,2 EDTA, 2 EGTA, and 10 DTT). Columnswere washed with equilibrating buffer, and PKC activity waseluted by means of a linear NaCl gradient (O-O.3 M) in buffer B. PKC eluates from DEAE-cellulose chromatography were assayedfor PKC as previously described(18). PKC activity wasdeterminedby measuring the transfer of 32P from [T-~~P]ATP to histone III-S. The final reaction mixture (240 ~1) contained 0.7-1.2 x lo6 counts/min [y- 32P]ATP 15 x 10m7 M ATP, 0.46 mM CaCl, 6.9 mM MgC12, and 3O’mg histone III-S. Each fraction was assayedin the presenceand absenceof phosphatidylserine (80 pg) and diolein (4 pg), added as a lipid suspensionin 100 mM Tris HCl, pH 7.5. The reaction was carried out at 30°C for 10 min and wasstoppedby adding 2 ml of 12% trichloroacetic acid (TCA) in the presenceof 600 pg y-globulin added as carrier. After centrifugation the pellet was dissolved in 0.5 ml 1 N NaOH and precipitated again with 12% TCA. After a second centrifugation the protein precipitate was dissolved in 1 N NaOH and 32P incorporation was measuredby scintillation counting in Ultima Gold (Packard). Measurement of [Ca2+ji. [Ca2+]i was measuredin confluent monolayerswith the fluorescent Ca2+probe fura-2/AM, adapting the method describedelsewhere(4). Cells were grown on glasscover slips in DMEM with 2% FCS. When confluencewas attained, the cells were washed twice in Krebs-Ringer buffer (KRB). The monolayerswere then covered with 400 ~1of KRB containing 2 PM fura- and 0.5% bovine serumalbumin (BSA) and wereincubated for 30 min at 37°C. At the end of the loading period, the slideswere washedtwice in KRB without albumin and inserted into glasscuvettes (1 x 1 cm) then placed in the thermostated holder of an LS-3 Perkin-Elmer fluorescence spectrophotometer. Continuous stirring was achieved with a magnetic stirrer. Fluorescenceof fura-2-loaded monolayerswas measuredusing excitation wavelength 339 nm and emission wavelength 505 nm. Calibration of the signal was performed with ionomycin and MnC12in the presenceof an excessof Ca2+, as reported elsewhere(4). Functional tests. Aortic smooth muscle cells seeded into 90-mm Petri or 6-well tissue culture plates were washedand incubated for 30 min at 37°C with 10 ml or 1 ml, respectively, with KRB buffer containing 0.2% BSA and 0.2% glucose.The supernatant was then replaced with fresh buffer, and the cells were incubated at 37°C with 95% air-5% CO, in the presenceof various stimuli. At the end of the incubation period, the prostacyclin content of the media was determined by a specific radioimmunoassayof its stable metabolite 6-keto-PGF,,,, as previously described (18). The cross-reactivity of the anti-6keto-PGF,,, rabbit antiserum was 14% with PGF1,,, 2% with PGE,, 1.7% with PGF,,,, and ~1% with the other major arachidonic acid metabolites.The intra- and interassaycoefficients of variation of the assay were 4 and 8%, respectively, and the sensitivity of the assaywas 3 pg. Statistical analysis. Student-Fisher unpaired bilateral t tests, analysis of variance using the Scheffe F test criterion for unbalanced groups, and principal component (factor) analysis by the method of least-squaresfit were usedwhere applicable. The data represent meanst SE of at least three experiments performed in duplicate or triplicate determinations.
IN AORTIC
SMOOTH
MUSCLE
E801
CELLS
l
RESULTS
Influence of PKC activation and [Ca2+]i elevation on prostacyclin production in aortic smooth muscle cells. Fig-
ure 1 shows the effect of maximal concentrations of AVP, A23187, and PMA on PKC activity and on prostacyclin production. Incubation with AVP (10m7 M) for 30 min enhanced prostacyclin production from 0.2 t 0.02 to 4.1
membranes 0
Basal
m
A23187
m
PMA A23187
0
non-treated
+ PMA
AVP
48h PMA
Fig. 1. Effect of A23187 and arginine vasopressin (AVP) on protein kinase C (PKC) activity and prostacyclin production in intact and PKC-depleted aortic smooth muscle cells. Aortic smooth muscle cells were pretreated with 100 nM PMA for 48 h and then incubated for 30 min with A23187 (5 X lOA M) or AVP (10s7 M). Prostacyclin production (A) and membranous and cytosolic PKC activity (B and C) were determined as described in MATERIALS AND METHODS. PKC activity is expressed as percentage of control values. Values are means t SE of 4 separate experiments. PMA, phorbol 12-myristate 13-acetate.
+ 0.3 ng/mg protein (P < 0.001, n = 3). The ionophore A23187 (5 X 10B6 M) increased prostacyclin formation to 3.5 t 0.2 ng/mg protein (P < 0.001, n = 3), whereas the PKC activator PMA (lo- 7 M) stimulated prostacyclin synthesis only to 1.0 t 0.1 ng/mg protein (P < 0.001, n = 3). However, PMA potentiated the A23187-induced prostacyclin formation by 240 t 14% (P c 0.001, n = 3, vs. A23187 alone). Exposure to PMA (10m7 M) for 30 min enhanced basal membranous PKC activity by 209 t 26% (P < 0.001, n = 4), whereas cytosolic PKC activity was decreased by 73 t 5% (P < 0.001, n = 4). AVP ( 10S7 M) increased membranous PKC activity by 52 t 12% (P < 0.01, n = 3) but had no significant effect on cytosolic PKC activity. In contrast, A23187 (5 x 10S6 M) did not modify membranous or cytosolic PKC activity in control and PMA-stimulated cells (Fig. 1). To determine whether A23187 could induce membranous PKC association, which dissociates when the 10 mM EGTA present in our homogenization buffer is added (see MATERIALS AND METHODS), we further measured PKC activity in cytosolic and membranous fractions prepared in the absence of EGTA. When cells were homogenized in the absence of EGTA, A23187 (5 x 10B6 M) appeared to induce a small but statistically insignificant increase in membranous PKC activity (Table 1).
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E802
REGULATION
OF PGIz
PRODUCTION
Table 1. Effect of EGTA on cytosolic and membranous PKC association in homogenates of A23187and PMA-stimulated cells Protein Treatment
Control A23187 PMA
+lO
mM
Kinase
C Activity,
cpm/mg
EGTA
protein
Without
EGTA
Cytosol
Membranes
Cytosol
Membranes
3,673+375 3,530&414 561k84
378t48 411t55 1,454+156
2,034+427 2,641&461 488t55
714t64 926&103 1,416+111
Values are means t SE of 4 separate experiments. Aortic smooth muscle cells were incubated for 30 min with phorbol 12-myristate 13acetate (PMA, 1O-7 M) or A23187 (5 x lo-(’ M) and membranous and cytosolic cell fractions were prepared in presence or absence of 10 mM EGTA. Protein kinase C (PKC) activity was determined as described in MATERIALS AND METHODS. PKC activity is expressed in counts per into histone III-S per 10 min per minute (cpm) of :32P incorporated milligram protein homogenate.
Long-term exposure to phorbol ester is known to desensitize PKC activity (26). In aortic smooth muscle cells, prolonged preincubation for 48 h with PMA (10M7 M) completely suppressed PKC activity as follows: it was no longer possible to detect any PKC activity, neither in control nor in AVP- or PMA-stimulated cells (Fig. 1). In these PKC-depleted cells, PMA did not stimulate prostacyclin production, whereas AVP-induced prostacyclin production was inhibited by 74 t 2% (P < 0.01, n = 3). In contrast, although A23187-induced prostacyclin production was not affected by PKC depletion, it was no longer potentiated by PMA. These observations suggest that, although an elevation of [Ca2+]i is a sufficient stimulus for prostacyclin production in these cells, PKC plays an important modulatory role during hormonal stimulation. This conclusion was further substantiated by the finding that staurosporine (5 x 10S7 M), which inhibited membranous and cytosolic PKC activity in both control- (85 and 64% inhibition, respectively) and PMA-stimulated cells (93 and 58% inhibition, respectively), did not inhibit A23187-induced prostacyclin production yet abolished the PMA-induced response in the presence and absence of A23187 (P < 0.005, n = 3, Fig. 2). Identical results were 0 I
q
C
IN AORTIC
SMOOTH
MUSCLE
CELLS
obtained with the highly selective PKC inhibitor CGP41251 (10e6 M, see Ref. 19), a compound that had no significant effect on the A23 187-stimulated response yet also inhibited the effect of PMA in the presence and absence of A23187 (P < 0.005, n = 4). To assess the effect of A23187 on cellular [Ca2+]i, we measured [Ca2+]i with the fluorescent probe fura-2/AM using the nonfluorescent derivative of A23187, 4-BrA23187. The addition of 4-Br-A23187 (5 x low6 M) resulted in an increase in [Ca2+]; from a resting value of 141 t 13 to 471 t 80 nM (n = 5) and lasting up to 3 min while AVP ( 10e7 M) induced a biphasic rise in [Ca2+]i, the transient [Ca”+]i spike recorded at 455 t 52 nM (n = 5) followed by a lower sustained phase as has previously been reported (see Ref. 6). Overall, 4-Br-A23187 was found to be a less potent stimulus for prostacyclin production than A23187. Maximal concentration of 5 X lo-” M 4-Br-A23 187 stimulated prostacyclin production from 0.12 t 0.02 to 0.45 t 0.06 ng/mg protein (P < 0.01, n = 3)) whereas A23 187 increased prostacyclin production to 1.30 t 0.03 ng/mg protein (P < 0.001, n = 3). Effect of short- and long-term PMA treatment on A23187- and A VP-induced prostacyclin production in rat aortic smooth muscle cells. Pretreating cells with 10S7 M
PMA resulted in a time-dependent potentiation of both the A23187- and the AVP-induced prostacyclin production, with maximal augmentations of 454 t 15 and 487 t 31% at 1 h, respectively (Fig. 3). Thus PKC activation by PMA appears to modulate AVP- and A23187-stimulated prostacyclin formation. Longer periods of preincubation with PMA resulted in a decrease of the PMA-induced potentiation. After 48 h of exposure to PMA, the A23187induced response was similar to that observed in nonpreincubated cells, whereas AVP-stimulated prostacyclin production was inhibited by 56 t 9% (Fig. 3). Prolonged effect of PKC activation on AVP- and A23187-induced prostacyclin production. Because PKC
activation is known to be responsible for the sustained phase of the biological response in many systems, we determined whether the PMA-induced activation of PKC and its potentiating effect on A23187- or AVP-induced
wlthout Inhibitor + staurosporine + CGP41251
A23
PMA
A23+PMA
Fig. 2. Effect of staurosporine and CGP-41251 on PMAand A23187induced 6-keto-PGF,,, production in aortic smooth muscle cells. Cells were stimulated for 30 min with PMA (10m7 M), A23187 (5 X 10ec’ M), or PMA and A23187 in absence or presence of staurosporine (5 X 10Y7 M) or CGP-41251 (lo-” M). 6-keto-PGF,(, production was determined as described in MATERIALS AND METHODS. Values are means * SE of 4 independent experiments.
0
1
2 Preincubation
3 24 with PMA (hours)
48
Fig. 3. Effect of PMA on A23187and AVP-induced prostacyclin production. Aortic smooth muscle cells were preincubated with 100 nM PMA for indicated times before being stimulated with A23187 (5 X lo-” M) or AVP (lOhY M) for 30 min. 6-Keto-PGF,,, production was determined as described in MATERIALS AND METHODS. Values are means + SE of 3-4 separate experiments.
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REGULATION
OF PGI,
PRODUCTION
prostacyclin formation persisted after the removal of PMA. In cells pretreated for 30 min with 10m7 M PMA, thus having PKC maximally activated (18), and then washed free of PMA, the response to the immediate addition of A23187 (Fig. 4B) or AVP (Fig. 4C) was potentiated by 548 t 109% (P < 0.01, n = 3) and 141 t 19% (P < 0.01, n = 3), respectively. This potentiating effect was still present 30 min after removal of PMA, before stimulation with A23187 or AVP (Fig. 4, B and C). After a l-h interval without PMA, the potentiating effect of PMA decreased to 256 t 50% of A23187-stimulated prostacyclin production in nonpretreated cells (P < 0.05 vs. A23187 alone, n = 3, Fig. 4B). Interestingly, 10m7 M PMA potentiated A23 187-induced prostaglandin release in cells previously pretreated with phorbol ester and allowed to rest for 60 min (Fig. 4, E vs. B), a response similar to that obtained in cells having been stimulated with A23187
IN AORTIC
SMOOTH
MUSCLE
E803
CELLS
immediately after 30 min of preincubation with PMA (Fig. 4B). In contrast to what was observed in A23187-stimulated cells, the potentiating effect of PMA pretreatment on the AVP-stimulated prostacyclin response was further increased after 60 min of incubation in the absence of PMA, reaching 363 t 33% of the AVP-induced response in nonpretreated cells (Fig. 4C). The presence of 10e7 M PMA during AVP stimulation did not further augment the AVP-induced response in these cells (Fig. 4F). When cells were preincubated with PMA for time periods of 5 or 10 min, which induce submaximal activation of PKC (6, 13), A23187-stimulated prostacyclin production was still augmented by 483 t 19 and 483 t 88%, respectively, compared with a potentiation of 1,405 t 41% in cells pretreated for 30 min. (Fig. 5). These weaker potentiating effects could still be observed after 30 min of incubation without PMA, before stimulation with A23187 (Fig. 5). In contrast, preincubation of cells with A23187 (5 x lo- ci M) for 30 min had no potentiating effect on PMA-induced prostacyclin production, even when stimulation occurred immediately after the end of A23187 pretreatment (data not shown). Relation between PKC activation and prostacyclin production. To investigate whether the persistence of the
effect of PMA on prostacyclin production 15-30 min after removal of PMA was due to persistent PKC activation, we compared membranous PKC activity with A23187-induced prostacyclin formation. Figure 6 shows that there was no strong correlation between membranous PKC activity and the potentiating effect of PMA pretreatment on A23187-induced prostacyclin formation. ‘, ,’
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-
0
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Fig. 4. Effect of various time intervals between initial PMA stimulation and addition of A23187 or AVP on subsequent prostacyclin response. Aortic smooth muscle cells were preincubated with PMA (lo-’ M) for indicated times. Medium was then removed and replaced by fresh Krebs-Ringer buffer (MATERIALS AND METHODS). Cells were either incubated immediately with A23187 (5 x lo-(’ M) or AVP (10Y7 M) for 30 min in presence (hatched bars) or absence (open bars) of PMA ( 10Y7 M) or were allowed to recuperate for indicated times before stimulation. Values are means t SE of 3-4 separate experiments. A: control. B: A23187 (5 x lo-(; M). C: AVP (1O-7 M). D: PMA (1OW M). E: A23187 (5 x lo-(; M) + PMA (lo+ M). F: AVP (1O-Y M) + PMA (1O-7 M).
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Fig. 5. Effect of changes in duration of quent prostacyclin response induced by smooth muscle cells were pretreated with 30 min. Cells were then either stimulated 3O-min recuperation period in absence means k SE of 4 separate experiments.
30 min interval
PMA pretreatment on subseA23187 30 min later. Aortic PMA ( 10m7 M) for 0,5, 10, or immediately (left) or after a of PMA (right). Values are
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E804
REGULATION
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to be inversely correlated with A23 187-induced prostacyclin production (r = -0.95, P < 0.005, n = 5), suggesting that the level of cytosolic PKC activity is a robust indicator for the degree of activation of the phosphorylation cascade leading to the prostacyclin response. DISCUSSION
3
x -5 -
IN AORTIC
*
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100 80 60
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PMA 30min
.PMA 30mln
PMA 30mln
P;A 30mln
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Fig. 6. Relationship between PKC activity and A23187-induced prostacyclin production. Aortic smooth muscle cells were pretreated with PMA (10v7 M) for 30,60, or 90 min either continuously, or, after 30 min of PMA-pretreatment, cells were incubated for another 30 or 60 min without PMA. Cells were then either stimulated with A23187 (5 X lo-” M) for 30 min and prostacyclin production was measured (A), or membranous and cytosolic PKC activities were determined (B and C, respectively). 6-Keto-PGF,,r and PKC activity were measured as described in MATERIALS AND METHODS. PKC activity is expressed as percentage of control values. Values are means k SE of 4 separate experiments.
Membranous PKC activity was maximally increased, to 450 t 43% of control values after 30 min of PMA treatment. Longer exposures to PMA for 60 and 90 min decreased membranous PKC activity to 271 t 19 and 176 t 13%, respectively, of control values. In contrast, A23187induced prostacyclin production was similar in cells pretreated for 30 or 60 min with PMA, and a longer preincubation period of 90 min augmented the PMA potentiating effect by 26 t 5% (Fig. 6). When cells that had been exposed for 30 min to PMA were incubated for 30 and 60 min without PMA, membranous PKC activity was still increased to 409 t 38 and 282 t 54%, respectively (P < 0.001 and P < 0.01 vs. control; n = 4 and 6, respectively). However, the potentiating effect of PMA pretreatment on prostacyclin production was significantly decreased (P < 0.05 and P < 0.01; n = 3 and 4, respectively) when compared with that of cells immediately stimulated after PMA pretreatment (Fig. 6). Thus membranous PKC activity cannot be correlated with A23187-stimulated prostacyclin production. This suggests that the prolonged effect of PMA on A23187-induced prostacyclin production is not due to high levels of membranous PKC activity but to an event following PKC activation. In contrast, cytosolic PKC activity was found
Activation of PKC and intracellular calcium mobilization are known to be essential intracellular events for various physiological processes (14, 16, 21). To investigate the respective roles of [Ca2+]i and PKC in aortic smooth muscle cells during AVP stimulation, we first determined the influence of A23187-induced [Ca2+]i elevation on PKC activation. In the presence of 1.2 mM extracellular Ca2+, the nonfluorescent A23 187 analogue 4-Br-A23187 was shown to induce a similar but more sustained increase in [Ca2+]i compared with that observed with AVP. We did not observe a significant effect of A23187 on membranous and cytosolic PKC activity in control or in PMA-stimulated aortic smooth muscle cells. Studies on the influence of A23187 on PKC activity are controversial. A23187 was found to increase membranous PKC activity in leukocytes (7) and in pinealocytes (14). It was also shown to enhance the binding of phorbol12,13-dibutyrate ([3H]PDB) to phagocyte (8), platelet (25), and leukemia cell (30) membranes, a result that is considered to correspond to an increase in membranous PKC activity. However, Saitoh et al. (25), studying the influence of epinephrine and A23187 on both [3H]PDB binding to membranous PKC and phosphorylation of a 47-kDa protein (a PKC-specific substrate) in platelets, have observed that epinephrine increased both [3H]PDB binding to membranous PKC and 47-kDa protein phosphorylation. In contrast, although A23187 increased [“H]PDB binding to membranous platelet PKC, it did not affect phosphorylation of the 47-kDa protein. Thus an increase in the [“H]PDB binding to membranous PKC does not always appear to reflect fully activated PKC. Various types of PKC association with cellular membranes have been suggested as follows: one that is entirely Ca2’ dependent and dissociable by the addition of EGTA, one that is induced by an increase in diacylglycerol and [Ca2*]i, and one that is induced by phorbol esters in the absence of an increase in [ Ca2+] i (11, 14). An elevation of [ Ca2+] i could induce PKC redistribution from the cytosol to the-membrane but would be insufficient to fully activate the membrane-bound PKC. The presence of diacylglycerol or phorbol esters, such as PMA, appears to be necessary to promote PKC penetration in membranous phospholipids to induce the conformational change resulting in a fully active lipid-protein complex (9). In addition, Christiansen et al. (7) have reported that, in platelets, the increase in [Ca2’]i has to be very high to induce the subcellular redistribution of PKC. Ionomycin, which increases [Ca2*]; to 2 mM in these cells, also induces the translocation of PKC to the membranous fraction, whereas N-formylmethionylleucylphenylalanine, which only increases [Ca2*]; to 500 nM, has no effect on membranous PKC activity (7). Thus the A23187-stimulated rise in [Ca2+]i (from 14 t 13 to 471 t 80 nM) observed in aortic smooth muscle cells could well be insufficient for
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REGULATION
OF PGI,
PRODUCTION
PKC activation in these cells. Nevertheless, A23187 was found to be a potent stimulus for prostacyclin production in aortic smooth muscle cells. Its stimulating action was found to be independent of PKC activation, since it was not affected by the PKC inhibitor staurosporine or by prolonged treatment (48 h) with PMA resulting in PKC depletion. These observations suggest that [ Ca2+]i itself plays an important role in prostacyclin production in aortic smooth muscle cells, probably by activating a calcium-sensitive phospholipase. In contrast, the PMA-induced potentiation of A23187induced prostacyclin release was abolished by both staurosporine and the highly selective PKC inhibitor CGP41251; it was also no longer present in PKC-depleted cells. In these cells, AVP-stimulated prostacyclin production was furthermore decreased by 70% when compared with nonpretreated cells. These results indicate that PKC plays a crucial modulatory role during hormonal stimulation of prostacyclin production in aortic smooth muscle cells. They are confirmed by a study of Pirotton et al. (23) who described a decrease of ATP- but not of A23187induced [ 3H] choline release in bovine aortic endothelial cells treated for 24 h with PMA. Kanterman et al. (15) observed that downregulation of PKC with long-term (24 h) PMA treatment in dopamine receptor-transfected ovary cells not only abolished the dopamine-induced stimulatory effect but also partially decreased the A23187-stimulated arachidonic acid release (15). The potentiating influence of PKC activation on A23 187-induced prostacyclin production persisted for -30 min after the removal of PMA, whereas in AVPstimulated cells, it was still present 1 h after the removal of PMA. This difference can be explained by the fact that A23187 induces only an elevation of [Ca2+]i without affecting PKC activity, whereas AVP acts through phosphatidylinositol4,5-bisphosphate hydrolysis, thus elevating both [ Ca2+]i and membranous PKC activity. Previous studies (28) have shown that angiotensin II (ANG II)induced PGI, production is potentiated by a previous exposure to AVP. This potentiating effect persists for 90 min after the removal of AVP, suggesting that a postreceptor event such as PKC activation is implicated. Barrett et al. (2) have shown that previous activation of adrenal glomerulosa cells with ANG II potentiates a second ANG II-induced aldosterone secretion even after a time interval of 35-60 min in the absence of ANG II. They noticed that this potentiating effect persisted only when the first stimulation with ANG II was long enough (20 min) to maximally stimulate PKC activity. In contrast, the potentiating effect of PMA on aortic smooth muscle cells persists also when the cells have been incubated for time periods (5-10 min) that do not allow for full PKC activation. The use of different stimuli could explain the discrepancy between our results and those of Barrett et al. (2). In summary, our determinations of PKC activity and prostacyclin production in aortic smooth muscle cells suggest that the “memory” effect of PMA-induced PKC activation is not due to sustained levels of membranous PKC activity but is probably linked to the phosphorylation of protein(s) after PKC activation.
IN AORTIC
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MUSCLE
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CELLS
We thank C. Gerber-Wicht, M. Klein, and M. Rey for excellent technical assistance and Dr A. M. Capponi for helpful discussion. This study was supported by Grant 31.21727.89 from the Swiss National Science Foundation. D. J. Church was further supported by a postgraduate scholarship from the Glaxo Institute of Molecular Biology, Geneva, Switzerland. Address for reprint requests: U. Lang, Div. of Endocrinology, Geneva Univ. Hospital, 24, Rue Micheli-du-Crest, CH-1211 Geneva 4, Switzerland. Received
29 October
1991; accepted
in final
form
22 May
1992.
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E806 (Abstract).
REGULATION
OF PGI,
PRODUCTION
IN AORTIC
Proc. Annu. Meet. Endow. Sot. 11th Washington DC
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