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Phorbol ester enhances activation of adenylate cyclase in Bovine aortic endothelial cells
Life Sciences, Vol. 54, pp. 87-94 Printed in the USA
Pergamon Press
PHORBOL ESTER ENHANCES ACTIVATION O F ADENYLATE CYCLASE IN BOVINE AORTIC ENDOTHELIAL CELLS D.C. Lefroy1, L.E. Donnelly, J.R. McEwan, J. MacDermot. Department of Clinical Pharmacology, Royal Postgraduate Medical School, Du Cane Road, London W12 0NN, UK. (Received in final form October 25, 1993) Summary Endothelial cells possess ll-adrenoceptors linked to adenylate cyclase which may regulate several aspects of endothelial cell function. The potential for this second messenger system to be modulated by protein kinase C activity was investigated. Bovine aortic endothelial cells (BAECs) were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Basal and forskolin-, sodium fluoride (NaF)-, and isoproterenol-stimulated adenylate cyclase activity was measured in homogenates from BAECs. fl-adrenoceptor density on membranes from BAECs was measured by l~Iiodocyanopindolol binding. Sodium dodecylsulfate-polyacrylamidegel electrophoresis of immunoprecipitated proteins was used to identify phosphorylated proteins. Pretreatment of BAECs with 100 nM PMA for 30 min increased basal adenylate cyclase activity above control levels, and also increased enzyme activity stimulated by forskolin, NaF, or isoproterenol. Pretreatment of BAECs for 60 min with 100 nM staurosporine, an inhibitor of protein kinase C, prevented the enhancement of adenylate cyclase activity caused by PMA. Treatment of BAECs with PMA did not trigger phosphorylation of the inhibitory guanine nucleotide-binding protein, and there was no change in BAEC B-adrenoceptor density following PMA pretreatment. Exposure of BAECs to ATP or bradykinin did not mimic the effects of phorbol ester. In conclusion, activation of protein kinase C by PMA enhanced adenylate cyclase activity in BAECs. However, ATP and bradykinin which activate endothelial cell surface receptors linked to phospholipase C did not mimic this effect.
Endothelial cell function is regulated by ceil-surface receptors which mediate responses through several transduction pathways. For example, receptors for bradykinin (1), histamine (2), thrombin (3), and ATP (4) activate phospholipase C. Activated phospholipase C hydrolyses membrane phosphatidylinositol 4,5-bisphosphate and releases inositol 1,4,5-trisphosphate, which causes a rise in intracellular free calcium (5) and in turn leads to release of endothelium-derived relaxing factor and prostacyclin. There is a parallel rise in diacyiglycerol which activates protein kinase C, an effect which is mimicked by phorbol esters (6). Activation of protein kinase C in endothelial cells by phorbol esters has many reported consequences, including alteration of cell morphology (7), increasing endothelial permeability (8), expression of leukocyte adhesion molecules (9), and increased angiotensin converting enzyme synthesis (10). Wagner et al (11) demonstrated a catecholamine-stimulated adenylate cyclase in capillary endothelium, and suggested that it
1 Correspondence should be addressed to: Dr David Lefroy, Cardiology, Hammersmith Hospital, Du Cane Road, London WI2 0HS, UK. Tel: 44 81 743 2030; Fax 44 81 740 8373 0024-3205/94 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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may regulate endothelial function (11). Subsequently, B-adrenoceptors (12), A2 purinoceptors (13), and calcitonin gene-related peptide receptors (14) that are linked to adenylate cyclase have been identified. Elevated cyclic AMP levels in endothelial cells have several effects, which include regulation of angiotensin converting enzyme activity (10), inhibition of secretion of tissue-type plasminogen activator (15), and inhibition of bradykinin-stimulated prostacyclin release (16). There is now good evidence that these distinct intracellular signalling pathways may interact at several levels and that protein kinase C has a central role in modulating their activities (17). In order to evaluate the possibility that such interactions occur in the vascular endothelium, we studied the effects of direct activation of protein kinase C by phorbol ester on adenylate cyclase activity in cultured endothelial cells.
Methods Reagents: [3H]-cyclic AMP (23 Ci.mmoll), [a32P]-ATP (10 - 50 Ci.mmol~), [-~2P]-H3PO4 (700 950 Ci.mmol-~), and [lZ~l]-iodocyanopindolol( - 2 0 0 0 Ci.mmol -~, ~zsICYP) were obtained from Amersham International plc, UK, and [3:P]-NAD (30 Ci.mmol 1) from New England Nuclear. Staurosporine was a gift from Dr K Scheibli, Ciba-Geigy, Switzerland. Ro20-1724 was a gift from Hoffman-La Roche. Streptomycin and penicillin were from Flow Laboratories, gentamicin from Gibco and 2-methylthio-ATP from Research Biochemical Inc, Natick, USA. Antisera with specificity for the C-terminal decapeptide of the ct subunits of the stimulatory (Gsa) and inhibitory (Gic¢) guanine nucleotide-binding proteins were obtained from Biomac Ltd, UK. A second anti-Gict antiserum (1010) was prepared in collaboration with IDRL, Department of Immunology, University of Birmingham, UK. The antibody was raised by immunisation of a sheep with the peptide [AAERSKMIDKNLREDGEKAYA1. All other reagents were obtained from Sigma Chemical Co. Ro20-1724 was dissolved in ethanol, and J~ICYP in methanol. Phorbol 12-myristate 13-acetate (PMA) and staurosporine were dissolved in dimethylsulfoxide (Me:SO). Cell culture: AG04762 Bovine aortic endothelial cells (BAECs) were obtained from the cell repository of the National Institute of Aging, USA. BAECs were cultured in Dulbecco's modified Eagle's medium, containing 10% fetal calf serum, 1.5 mM glutamine, 50 izg.m1-1 gentamicin, 25 U.m1-1 penicillin, and 25 ~tg.m1-1 streptomycin. Culture flasks were maintained at 37°C in a humidified atmosphere of 5% CO: and 95% air. BAECs were passaged with 0.5 mg.ml -~ trypsin and 0.25 mM EDTA, and used at confluence at passages 15 - 20. Where indicated, the culture medium was changed for fresh serum-free culture medium containing PMA, staurosporine, ATP, bradykinin, or 0.87 mM Me, SO carrier alone before harvesting. BAECs were washed once and harvested in Dulbecco's phosphate-buffered saline (without Ca2+ or Mg :+) using a rubber scraper. Cells were pelleted at 650g for 4 min and stored at -80°C. Homogenates for measurement of adenylate cyclase activity were prepared at 4°C in 25 mM Tris-HCl buffer pH 7.4, containing 0.29 M sucrose and 0.25 mM EDTA, by disruption of cells with 30 strokes of a tightly fitting Dounce homogenizer. Membranes for ~:-~ICYPbinding measurements were prepared at 4°C in 50 mM Tris-HCl buffer pH 7.4, containing 0.25 mM EDTA, by disruption of cells with 30 strokes of a Dounce homogenizer. Membranes were pelleted at 160,000g for 30 min, and then resuspended in the same buffer. Membranes were washed three times before use. The protein content ofhomogenates and membranes was determined by the method of Lowry et al (18). Adenylate cyclase activity: Adenylate cyclase activity was measured in BAEC homogenates by a modification of the method of Salomon et al (19). Determinations were performed in triplicate at 37°C in 50 mM Tris-HC1 buffer pH 7.4, 87 mM sucrose, 5 mM Mg acetate, 1 mM cyclic AMP, 4 #M GTP, 20 mM creatine phosphate, 10 IU creatine kinase, 0.25 mM Ro20-1724 (a cyclic AMP-specific phosphodiesterase inhibitor), 1 mM [et32p]-ATP (3 ~tCi/reaction mixture), and 20 - 30 tzg homogenate protein in a reaction volume of 100 #1. Where indicated, either forskolin, sodium fluoride (NaF) or isoproterenol was also added, The reaction was terminated after 20 min by the addition of 800/~1 6.25% trichloroacetic acid, and a trace amount of [3H]-cyclic AMP (approximately 10,000 c.p.m.) was also added at this stage. ATP and cyclic AMP were separated chromatographically using Dowex and alumina
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columns, and the percentage fractional recovery of cyclic AMP (50 - 90%) was determined from the recovery of [3H]-cyclic AMP. Adenylate cyclase activity (pmol cyclic AMP.minLmg t protein) was derived from the 32p activity associated with the cyclic AMP fraction in the eluate from the columns which was then corrected for the fractional recovery of [3H]-cyclic AMP. l~-Adrenoceptor bindinz: I~ICYP binding to BAEC membranes was determined in triplicate samples at 25°C in the presence of 10/~M phentolamine, 5 mM MgCI:, 50 mM Tris-HCl buffer pH 7.4, selected concentrations (5 - 500 pM) of I~ICYP, and 10 - 20/~g membrane protein in a reaction volume of 200 ~d, and in the absence or presence of 50 ~tM isoproterenol to determine non-specific binding. After 2 h, membrane-bound activity was separated by negative pressure filtration through GF/C glass fiber filter paper (Whatman) using a Brandel M-24R cell harvester. Filters were washed three times with 4 ml 50 mM Tris-HCl buffer pH 7.4 with 5 mM MgC12 at 4°C, and filter-bound activity was counted on an LKB-WalIac Clinigamma 1272-004 gamma counter at 60% efficiency. Filter-bound activity was less than 10% of total radioactivity of the reaction mixtures. In preliminary experiments, binding was shown to saturate ( > 90%) within 2 h, and was linear with respect to protein within the range 10 - 50/zg. Identification of phosphorylated protein: In experiments to identify phosphorylated Gia, BAEC cultures were washed three times with phosphate-free culture medium, and were then cultured in phosphatefree culture medium in the presence of [32p]-H3PO4 0.2 mCi.m1-1 for 3 h. Either 100 nM PMA or 0.87 mM Me2SO was then added to the culture medium for the final 30 rain before harvesting. The cells were harvested, pelleted and stored as described above. Gic~ was isolated from the 32p-labeled cells by immunoprecipitation as described by Anderson and Blobel (20). Cell pellets were solubilised by boiling in 50 Izl 0.1% sodium dodecylsulfate (SDS) and then added to 200 #1 dilution buffer (1.25% Triton X-100, 190 mM NaCI, 60 mM Tris-HCl buffer pH 7.4, 6 mM EDTA). A mixture of 2 specific anti-Giot antisera (24/~1) was then added, and the mixture allowed to stand at 4°C for 18 h before centrifugation at 10,000g for 2 rain. The supernatant was added to 75 izl of a 1:1 suspension of protein A-sepharose CL-4B with continuous mixing for 2 h at 37°C. The sepharose beads were pelleted by centrifugation for 10 s in a microcentrifuge, and washed 4 times with washing buffer (0.1% Triton X-100, 0.02% SDS, 150 mM NaCI, 50 mM Tris-HCl buffer pH 7.5, 5 mM EDTA) before a final wash in the same buffer without detergent. The supernatant was removed, and 80/A sample buffer (625 mM Tris-HCl buffer pH 6.8, 10% glycerol, 1% SDS, 1% g-mercaptoethanol, 0.01% bromo-phenol blue dye) was added to the beads and boiled for 3 min. The proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and 3:p_ labeled proteins identified by gel autoradiography. Data analysis: All data are expressed as mean + S.E.M. for separate experiments. Receptor binding studies were analyzed with a non-linear iterative curve-fitting program (GrafPAD Inplot, Grafpad Software, San Diego, USA). Student's t test was used for comparisons between treatments, except where indicated when two way analysis of variance (ANOVA) was used. A p value < 0.05 was considered statistically significant.
Results Effect of PMA on adenylate cyclase activity in BAECs: BAECs were cultured in the absence or presence of 100 nM PMA for 30 min before harvesting. Cell homogenates were made, and adenylate cyclase activity was measured in the absence or presence of either 10/~M forskolin, which stimulates the adenylate cyclase catalytic subunit directly (21), or 10 mM NaF, which stimulates adenylate cyclase via the stimulatory G protein (Gs) (22), or 10/xM isoproterenol, (Fig. 1). Homogenates from cells pretreated with PMA showed significantly increased basal and stimulated adenylate cyclase activity compared with control (Fig. 1). BAECs were cultured in the absence or presence of selected concentrations (0.1 - 100 nM) of PMA for 30 min before harvesting (Fig. 2a). The enhancement of basal, 10 ~tM forskolin-, and 10 mM NaF-
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stimulated adenylate cyclase activity in cell homogenates increased with PMA concentration throughout this range (Fig. 2a). Isoproterenol (10 #M)-stimulated adenylate cyclase activity was only enhanced following exposure to the highest concentration of PMA (100 nM). BAECs were also cultured for selected times (0 - 180 min) in the presence of 100 nM PMA (Fig. 2b). The greatest enhancement of basal and stimulated adenylate cyclase activity was observed at 15 - 30 min, and had diminished towards control values by 180 min (Fig. 2 b ) . 150 ~o o
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Fig. 1 Effect of PMA pretreatment on adenylate cyclase activity in BAEC homogenates. BAECs were cultured in the absence (open bars) or presence (hatched bars) of 100 nM PMA for 30 min before harvesting. Basal and forskolin (Forsk, 10 p.M)-, NaF (10 raM)-, or isoproterenol (Isop, 10/~M)stimulated adenylate cyclase activity was measured in BAEC homogenates. Results show the mean + S.E.M. of 14 similar experiments, ** p < 0.01 compared with untreated BAEC homogenates. ~
a 200
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Fig. 2 Concentration and time-dependence of the effect of PMA on adenylate cyclase activity in BAECs. (a) BAECs were cultured in the presence of selected concentrations of PMA for 30 min before harvesting. (b) BAECs were cultured in the presence of 100 nM PMA for selected times before harvesting. Basal (O) and forskolin (10/~M, A)-, NaF (10 mM, II)- and isoproterenol (10 #M, * )-stimulated adenylate cyclase activity was then measured in BAEC homogenates. Each point represents the mean _+ S.E.M. of triplicate determinations and the results are a representative example of 3 similar experiments. Concentration-response relationships were determined for forskolin, NaF, and isoproterenol in homogenates from cells cultured in the absence or presence of 100 nM PMA (Fig. 3). PMA pretreatment enhanced adenylate cyclase activity at all concentrations of forskolin (0. ! - 100 p.M), NaF (1 - 100 mM), or isoproterenol (0.04 - 4 #M) (I9 < 0.0001, two way ANOVA, Fig. 3). Low concentrations of NaF (1 10 mM) activated adenylate cyclase (Fig. 3b) (22); at higher concentrations (40 - 400 mM), an inhibitory effect of NaF upon adenylate cyclase activity was observed (Fig. 3b). A similar 'bell-shaped'
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concentration-response curve with inhibition of adenylate cyclase at high concentrations of NaF ( > 10 mM) has been observed previously in cultured NG108-15 neuroblastoma cells (23), and this most probably reflects activation of the inhibitory guanine nucleotide-binding protein (Gi) as well as inhibition mediated by a Gi-independent mechanism (23).
•~ 150
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Fig. 3 Concentration-response curves for adenylate cyclase activity in BAEC homogenates stimulated by (a) forskolin, (b) NaF and (c) isoproterenol. BAECs were cultured in the absence (©) or presence ( e ) of 100 nM PMA for 30 min before harvesting. In (c), curves were fitted according to a onesite model. Each point represents the mean + S.E.M. of triplicate determinations and the results are a representative example of 3 similar experiments. a b
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Fig.4 Effect of staurosporine pretreatment on adenylate cyclase activity in BAEC homogenates. (a) BAECs were cultured in the absence (open bars) or presence (hatched bars) of 100nM stanrosporine for 60rain before harvesting. Basal and forskolin (Forsk, 10 #M)-, NaF (10 raM)-, or isoproterenol (Isop, 10 #M)-stimulated adenylate cyclase activity was measured in BAEC homogenates. Results are the mean + S.E.M. of 6 experiments. (b) BAECs were cultured in the absence or presence of 100 nM staurosporine for 30 min and then for a further 30 min following the addition of Me2SO or 100 nM PMA to the culture medium before harvesting. Open bars: MezSO alone; stippled bars: PMA alone; solid bars: staurosporin followed by PMA. Results are the mean _+ S.E.M. of 3 experiments, * p < 0.05 compared with BAECs pretreated with PMA alone. Effect of staurosporine: BAECs were cultured in the absence or presence of 100nM staurosporine (an inhibitor of protein kinase C) for 60 min before harvesting (Fig. 4a). Basal and forskolin (10 t~M)-, NaF (10 mM)-, and isoproterenol (10 ~tM)-stimulated adenylate cyclase activity in cell homogenates was unchanged compared with control homogenates (Fig. 4a). BAECs were cultured in the absence or presence of I00 nM staurosporine for 60 min and then in the absence or presence of 100 nM PMA for 30 min before harvesting (Fig. 4b). Basal and forskolin-, NaF-, and isoproterenol-stimulated adenylate cyclase
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activity in homogenates from cells pretreated with staurosporine and PMA was significantly lower (p < 0.05 as indicated) than that in homogenates from cells pretreated with PMA alone, but was not significantly different from that in homogenates from untreated cells (Fig. 4b). Effect of PMA on 8-adrenoceptors on BAECs: Membranes were prepared from BAECs cultured in the absence or presence of 100 nM PMA for 30 min before harvesting. Specific ~z~ICYP binding to the membranes was measured. There was no significant difference of receptor affinity or number between membranes from the PMA-pretreated cells (Kd = 53.5 + 4.8 pM; B ~ = 144.5 + 18.6 fmol.mglprotein, n = 5) and membranes from control cells (Kd = 48.8 + 7.8 pM; B~,~ = 113.9 4- 13.6 fmol.mg-lprotein, n = 5). Effect of ATP or bradykinin on adenylate cyclase activity in BAECs: BAECs were cultured in the absence or presence of 50 #M ATP (n = 5), or 10/xM bradykinin (n = 3) for 30 min before harvesting. Basal, and forskolin (10 #M)-, NaF (10 mM)-, or isoproterenol (10 #M)-stimulated adenylate cyclase activity was measured. Adenylate cyclase activity was unaltered by pretreatment with bradykinin, although basal and stimulated enzyme activity was enhanced by pretreatment with ATP (basal 21.5 + 5.7 vs 19.1 4- 5.7, p = 0.01; forskolin 79.5 4- 11.4 vs 62.4 4- 4.63, p = 0.02; fluoride 93.3 4- 17.2 vs 73.3 4- 14.2, p = 0.008; isoproterenol 57.8 ___ 8.6 vs 43.7 + 7.0 pmol cAMP.min-Lmg~protein, p = 0.04). However, the increase mediated by ATP was not inhibited by pretreatment of BAECs with 100 nM staurosporine for 60 min before harvesting. Furthermore, the effect of ATP was not mimicked by fi,^/-methylene ATP (0.1 #M - 1 raM, n = 3) nor by 2-methylthio-ATP (10 nM - 100/zM, n = 3). Effect of PMA on Gic~ phosphorylation: Gi~ was immunoprecipitated from cell homogenates which had been treated for 30 min at 37°C with 20 #g.ml -~ pertussis toxin (preactivated by heating at 37°C for 30 min in the presence of 25 mM dithiothreitol) in the presence of 100 /zCi.m1-1 [3~-p]-NAD. In pilot experiments, there was inefficient immunoprecipitation of Gic¢ with a single Gi~ antiserum. However, the addition of two anti-Gi~ antisera, with specificity for different regions of the protein, resulted in immunoprecipitation of a single protein band at 46 kDa which was radiolabeled and corresponded to [32p]_ ADP-ribosylated Gi~. This served as a positive control for the immunoprecipitation. BAECs were labeled for 3 h with [32P]-H3PO4. Thereafter, 100 nM PMA (n = 5), or 0.87 mM Me2SO alone (n = 5), was added to the cultures tbr a further 30 min. There was no evidence of phosphorylation of Gic~ under these conditions. In similar experiments, BAEC homogenates were treated for 30 min at 37°C with 100 #g.m1-1 cholera toxin (preactivated according to the protocol for pertussis toxin) in the presence of 100 p.Ci.m1-1 [32p]-NAD. Following immunoprecipitation with anti-Gsc~ antiserum, no radiolabeled protein was detected.
Discussion This study showed that pretreatment of AG04762 bovine aortic endothelial cells (BAECs) in culture with PMA, an activator of protein kinase C, led to modulation of adenylate cyclase so that basal, and forskolin-, NaF-, and isoproterenol-stimulated adenylate cyclase activity was enhanced (Fig. 1). The enhancement of adenylate cyclase activity by PMA was not seen in homogenates from BAEC cultures which had been pretreated with staurosporine, an inhibitor of protein kinase C, although staurosporine pretreatment alone had no effect on adenylate cyclase activity (Fig. 4). The effect of PMA on adenylate cyclase activity was present within 15 min of exposure and had decreased towards the untreated control values by 180 min (Fig. 2b). The rapidity of onset, and the concentration of PMA at which this effect occurred, suggest that it was mediated by phosphorylation of component(s) of the adenylate cyclase complex and not by altered rates of synthesis of these proteins. Adenylate cyclase activity was measured in cell homogenates and so the results presented here reflect true changes in activation of adenylate cyclase, and not, for example, changes in phosphodiesterase activity, which might complicate similar experiments where cyclic AMP concentration is determined in intact cells.
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Phorbol esters which activate protein kinase C modulate adenylate cyclase activity in several different cell lines (17). Depending on the cell type used, adenylate cyclase activity is increased or decreased, and phorbol ester-induced modulations of receptor sensitivity (24-26), Gs activity (27), Gi activity (28-30), and catalytic subunit phosphorylation (31) have been observed. In our study, the major effect was stimulatory leading to enhanced basal and forskolin- and NaF-stimulated adenylate cyclase activity (Fig. 3), and was presumably mediated either through Gs (increased activity), Gi (reduced activity) or the catalytic subunit of adenylate cyclase. Under the conditions of this study, Gs was present in too low a concentration to allow reproducible immunoprecipitation of Gsc~ in detectable quantities. Phosphorylation of Gsa thus remains a possible, but unlikely, mechanism by which PMA-stimulated enhancement of adenylate cyclase activity is mediated. Phosphorylation of Gsc~has never been demonstrated in intact cells, although modulation of Gso~ activity has been demonstrated in rat hepatocytes in response to protein kinase C activation (27). The mechanism of the alteration of Gs activity under these circumstances remained speculative (27). In contrast, phorbol ester-induced phosphorylation of Giot has been demonstrated in intact rat hepatocytes (29), and has been shown to be associated with reduced Gi activity and therefore increased adenylate cyclase activity. Although we were able to immunoprecipitate Gia from BAEC homogenates, there was no detectable PMA-induced phosphorylation of Gic~ under the conditions of this study. The enhancement by PMA of isoproterenol-stimulated adenylate cyclase activity in BAECs was less than that of forskolin-stimulated or NaF-stimulated adenylate cyclase activity (Fig. 1). This suggests the possibility that protein kinase C activation may have a separate and inhibitory effect on the t~-adrenoceptor. AG04762 BAECs possess f~- and ll2-adrenoceptors in approximately equal numbers, although adenylate cyclase activation is mediated predominantly via the 1~2-adrenoceptor subtype (Lefroy, unpublished observations). Others (32,33) have found that phorbol esters modulate fi-adrenoceptor function by receptor phosphorylation, and that there is a potential site for protein kinase C-mediated phosphorylation on the third cytoplasmic domain of the fJ2-adrenoceptor. Phosphorylation at this site leads to reduced coupling to Gs (33) and reduced sensitivity to epinephrine-induced adenylate cyclase activation (32). A similar mechanism could account for our observation of reduced fi-adrenoceptor function in the absence of a change in receptor density, although the PMA-dependent changes in adenylate cyclase activation by NaF and forskolin imply an additional effect of protein kinase C on either the regulatory G-proteins or the catalytic subunit. In our experiments, the effect of PMA was not mimicked by ATP or bradykinin, although activation of endothelial cell surface receptors to these agonists has been shown to stimulate phospholipase C (1,4). We observed a stimulatory effect of ATP on adenylate cyclase activity. However, both the lack of a similar effect mediated by selected ATP analogs, and also the failure of staurosporine to reverse the effect, suggested that this effect of ATP was not mediated by P2v-purinoceptors, and was independent of protein kinase C. It is possible that this effect of ATP was mediated by P2u-purinoceptors which have recently been demonstrated on BAECs (34). Alternatively, ATP may act through phosphorylation of a cell surface substrate by an extracellular (ecto-) kinase, but this mechanism is reportedly very sensitive to inhibition by staurosporine (35), and is therefore unlikely to be the explanation for our observation. Cultured BAECs have been widely used as a model system for evaluating endothelial function and after repeated passaging they maintain a stable phenotype which closely resembles that of primary endothelial cell cultures. However, there are important differences in function of endothelium derived from different sources such as large vessels compared with the microvasculature (36), and it remains to be established whether interaction between protein kinase C and adenylate cyclase activity is a feature common to all endothelial cell types. In conclusion, our data show that activation of protein kinase C in BAECs by PMA modulates adenylate cyclase activity. The mechanism for this effect may involve separate alterations in activity of the fl-adrenoceptor, G-proteins or the catalytic subunit of adenylate cyclase. Neither ATP nor bradykinin mimicked this effect under the conditions of this study, although it is possible that, under physiological conditions during which there may be more sustained activation of protein kinase C, this effect might be significant. In BAECs, the potential exists for 'crosstalk' between distinct signal transduction pathways. Interaction between these signalling pathways may be important in the control of endothelial cell function.
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Acknowledgement This research was supported in part by a P r o g r a m m e Grant from the W e l l c o m e Trust. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
T.L. LAMBERT, R.S. KENT and A.R. WHORTON, J. Biol. Chem. 26_._.!15288-15293 (1986). W.W. LO and T.P. FAN, Biochem. Biophys. Res. Commun. 14847-53 (1987). E.A. JAFFE, J. GRULICH, B.B. WEKSLER, G. HAMPEL and K. WATANABE, J. Biol. Chem. 26__22 8557-8565 (1987). J-M. BOEYNAEMS and J.D. PEARSON, Trends Pharmacol. Sci. 1.~134-37 (1990). M.J. BERRIDGE, Nature 36_..!315-325 (1993). Y. NISHIZUKA, Nature 30_.._88693-698 (1984). A.S. ANTONOV, M.E. LUKASHEV, Y.A. ROMANOV, V.A. TKACHUK, V.S. REPIN and V.N. SMIRNOV, Proc. Natl. Acad. Sci. USA 8_..339704-9708 (1986). M.A. MURRAY, D.D. HEISTAD and W.G. MAYHAN, Circ. Res. 6....881340-1348 (1991). J.G. GENG, M.P. BEVILACQUA, K.L. MOORE, T.M. MC1NTYRE, S.M. PRESCOTT, J.M. KIM, G.A. BLISS, G.A. ZIMMERMAN and R.P. MCEVER, Nature 34__._~3757-760(1990). N. IWAI, M. MATSUNAGA, T. KITA, M. TEl and C. KAWAI, Biochem. Biophys. Res. Commun. 14____99 1179-1185 (1987). R.C. WAGNER, P. KREINER, R.J. BARRNETT and M.W. BITENSKY, Proc. Natl. Acad. Sci. USA 6_..99 3175-3179 (1972). V. BOUNASSISI and J.C. VENTER, Proc. Natl. Acad. Sci. USA 7...331612-1616 (1976). J. LUTY, J.A. HUNT, P.K. NOBBS, E. KELLY, M. KEEN and J. MACDERMOT, Cardiovasc. Res. 2__33 303-307 (1989). D. CROSSMAN, J. MCEWAN, J. MACDERMOT, I. MACINTYRE and C.T. DOLLERY, Br. J. Pharmacol. 9__22695-701 (1987). R.B. FRANCIS JR and S. NEELY, Biochim. Biophys. Acta 1012207-213 (1989). H. PARSAEE, J.R. MCEWAN, S. JOSEPH, and J. MACDERMOT, Br. J. Pharmacol. 1071013-1019 (1992). M.D. HOUSLAY, Eur. J. Biochem. 19_..559-27 (1991). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and P.J. RANDALL, J. Biol. Chem. 193265-275 (1951). Y. SALOMAN, C. LONDOS and M. RODBELL, Anal. Biochem. 58541-548 (1974). D.J. ANDERSON and G. BLOBEL, Method. Enzymol. 9_._66111-120 (1983). K.B. SEAMON and J.W. DALY, Adv. Cyclic Nucleotide Res. 2.._01-150 (1986). P.C. STERNWEISS, J.K. NORTHUP, M.D. SMIGEL and A.G. GILMAN, J. Biol. Chem. 25___6.611517-11526 (1981). E. KELLY, M. KEEN, P. NOBBS and J. MACDERMOT, Br. J. Pharmacol. 10__.00223-230 (1990). A. YAMASHITA, T. KUROKAWA, T. DAN'URA, K. HIGASHI and S. ISHIBASHI, Biochem. Biophys. Res. Commun. 13__88125-130 (1986). T. MURAYAMA, Y. NOMURA and M. UI, J. Biol. Chem. 264 15186-15191 (1989). M.T. NAKADA, J.M. STADEL and S.T. CROOKE, J. Pharmacol. Exp. Ther. 25_..~3221-229 (1990). S.M.T. HERNANDEZ-SOTOMAYOR, M. MACIAS-SILVA, C.C. MALBON and J.A. GARCIA-SAINZ, Am. J. Physiol. 26__9_0C259-C265 (1991). T. KATADA, A.G. GILMAN, Y. WATANABE, S. BAUER and K.H. JAKOBS, Eur. J. Biochem. 151 431-437 (1985). N.J. PYNE, G.J. MURPHY, G. MILLIGAN and M.D. HOUSLAY, FEBS Lett. 24__.3377-82 (1989). J.A. GARCIA-SAINZ and G. GUTII~RREZ-VENEGAS, FEBS Lett. 25__.!7427-430 (1989). T. YOSHIMASA, D.R. SIBLEY, M. BOUVIER, R.J. LEFKOWlTZ and M.G. CARON, Nature 32"/67-70 (1987). J.A. JOHNSON, R.B. CLARK, J. FRIEDMAN, R.A.F. DIXON and C.D. STRADER, Mol. Pharmacol. 3.._88 289-293 (1990). M. BOUVIER, N. GUILBAULT and H. BONIN, FEBS Lett. 27_..99243-248 (1991). S. MOTTE, S. PIROTTON and J.M. BOEYNAEMS, Circ. Res. 7_22504-510 (1993). S. PIROTTON, O. BOUTHERIN-FALSON, B. ROBAYE and J-M. BOEYNAEMS, Biochem. J. 28.._55585-591 (1992). B.R. ZETTER, Biology of Et~othelial Cells, E.A. Jaffe (Ed), 14-26, Martinus Nijhoff Boston (1984).
Pergamon Press
PHORBOL ESTER ENHANCES ACTIVATION O F ADENYLATE CYCLASE IN BOVINE AORTIC ENDOTHELIAL CELLS D.C. Lefroy1, L.E. Donnelly, J.R. McEwan, J. MacDermot. Department of Clinical Pharmacology, Royal Postgraduate Medical School, Du Cane Road, London W12 0NN, UK. (Received in final form October 25, 1993) Summary Endothelial cells possess ll-adrenoceptors linked to adenylate cyclase which may regulate several aspects of endothelial cell function. The potential for this second messenger system to be modulated by protein kinase C activity was investigated. Bovine aortic endothelial cells (BAECs) were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Basal and forskolin-, sodium fluoride (NaF)-, and isoproterenol-stimulated adenylate cyclase activity was measured in homogenates from BAECs. fl-adrenoceptor density on membranes from BAECs was measured by l~Iiodocyanopindolol binding. Sodium dodecylsulfate-polyacrylamidegel electrophoresis of immunoprecipitated proteins was used to identify phosphorylated proteins. Pretreatment of BAECs with 100 nM PMA for 30 min increased basal adenylate cyclase activity above control levels, and also increased enzyme activity stimulated by forskolin, NaF, or isoproterenol. Pretreatment of BAECs for 60 min with 100 nM staurosporine, an inhibitor of protein kinase C, prevented the enhancement of adenylate cyclase activity caused by PMA. Treatment of BAECs with PMA did not trigger phosphorylation of the inhibitory guanine nucleotide-binding protein, and there was no change in BAEC B-adrenoceptor density following PMA pretreatment. Exposure of BAECs to ATP or bradykinin did not mimic the effects of phorbol ester. In conclusion, activation of protein kinase C by PMA enhanced adenylate cyclase activity in BAECs. However, ATP and bradykinin which activate endothelial cell surface receptors linked to phospholipase C did not mimic this effect.
Endothelial cell function is regulated by ceil-surface receptors which mediate responses through several transduction pathways. For example, receptors for bradykinin (1), histamine (2), thrombin (3), and ATP (4) activate phospholipase C. Activated phospholipase C hydrolyses membrane phosphatidylinositol 4,5-bisphosphate and releases inositol 1,4,5-trisphosphate, which causes a rise in intracellular free calcium (5) and in turn leads to release of endothelium-derived relaxing factor and prostacyclin. There is a parallel rise in diacyiglycerol which activates protein kinase C, an effect which is mimicked by phorbol esters (6). Activation of protein kinase C in endothelial cells by phorbol esters has many reported consequences, including alteration of cell morphology (7), increasing endothelial permeability (8), expression of leukocyte adhesion molecules (9), and increased angiotensin converting enzyme synthesis (10). Wagner et al (11) demonstrated a catecholamine-stimulated adenylate cyclase in capillary endothelium, and suggested that it
1 Correspondence should be addressed to: Dr David Lefroy, Cardiology, Hammersmith Hospital, Du Cane Road, London WI2 0HS, UK. Tel: 44 81 743 2030; Fax 44 81 740 8373 0024-3205/94 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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may regulate endothelial function (11). Subsequently, B-adrenoceptors (12), A2 purinoceptors (13), and calcitonin gene-related peptide receptors (14) that are linked to adenylate cyclase have been identified. Elevated cyclic AMP levels in endothelial cells have several effects, which include regulation of angiotensin converting enzyme activity (10), inhibition of secretion of tissue-type plasminogen activator (15), and inhibition of bradykinin-stimulated prostacyclin release (16). There is now good evidence that these distinct intracellular signalling pathways may interact at several levels and that protein kinase C has a central role in modulating their activities (17). In order to evaluate the possibility that such interactions occur in the vascular endothelium, we studied the effects of direct activation of protein kinase C by phorbol ester on adenylate cyclase activity in cultured endothelial cells.
Methods Reagents: [3H]-cyclic AMP (23 Ci.mmoll), [a32P]-ATP (10 - 50 Ci.mmol~), [-~2P]-H3PO4 (700 950 Ci.mmol-~), and [lZ~l]-iodocyanopindolol( - 2 0 0 0 Ci.mmol -~, ~zsICYP) were obtained from Amersham International plc, UK, and [3:P]-NAD (30 Ci.mmol 1) from New England Nuclear. Staurosporine was a gift from Dr K Scheibli, Ciba-Geigy, Switzerland. Ro20-1724 was a gift from Hoffman-La Roche. Streptomycin and penicillin were from Flow Laboratories, gentamicin from Gibco and 2-methylthio-ATP from Research Biochemical Inc, Natick, USA. Antisera with specificity for the C-terminal decapeptide of the ct subunits of the stimulatory (Gsa) and inhibitory (Gic¢) guanine nucleotide-binding proteins were obtained from Biomac Ltd, UK. A second anti-Gict antiserum (1010) was prepared in collaboration with IDRL, Department of Immunology, University of Birmingham, UK. The antibody was raised by immunisation of a sheep with the peptide [AAERSKMIDKNLREDGEKAYA1. All other reagents were obtained from Sigma Chemical Co. Ro20-1724 was dissolved in ethanol, and J~ICYP in methanol. Phorbol 12-myristate 13-acetate (PMA) and staurosporine were dissolved in dimethylsulfoxide (Me:SO). Cell culture: AG04762 Bovine aortic endothelial cells (BAECs) were obtained from the cell repository of the National Institute of Aging, USA. BAECs were cultured in Dulbecco's modified Eagle's medium, containing 10% fetal calf serum, 1.5 mM glutamine, 50 izg.m1-1 gentamicin, 25 U.m1-1 penicillin, and 25 ~tg.m1-1 streptomycin. Culture flasks were maintained at 37°C in a humidified atmosphere of 5% CO: and 95% air. BAECs were passaged with 0.5 mg.ml -~ trypsin and 0.25 mM EDTA, and used at confluence at passages 15 - 20. Where indicated, the culture medium was changed for fresh serum-free culture medium containing PMA, staurosporine, ATP, bradykinin, or 0.87 mM Me, SO carrier alone before harvesting. BAECs were washed once and harvested in Dulbecco's phosphate-buffered saline (without Ca2+ or Mg :+) using a rubber scraper. Cells were pelleted at 650g for 4 min and stored at -80°C. Homogenates for measurement of adenylate cyclase activity were prepared at 4°C in 25 mM Tris-HCl buffer pH 7.4, containing 0.29 M sucrose and 0.25 mM EDTA, by disruption of cells with 30 strokes of a tightly fitting Dounce homogenizer. Membranes for ~:-~ICYPbinding measurements were prepared at 4°C in 50 mM Tris-HCl buffer pH 7.4, containing 0.25 mM EDTA, by disruption of cells with 30 strokes of a Dounce homogenizer. Membranes were pelleted at 160,000g for 30 min, and then resuspended in the same buffer. Membranes were washed three times before use. The protein content ofhomogenates and membranes was determined by the method of Lowry et al (18). Adenylate cyclase activity: Adenylate cyclase activity was measured in BAEC homogenates by a modification of the method of Salomon et al (19). Determinations were performed in triplicate at 37°C in 50 mM Tris-HC1 buffer pH 7.4, 87 mM sucrose, 5 mM Mg acetate, 1 mM cyclic AMP, 4 #M GTP, 20 mM creatine phosphate, 10 IU creatine kinase, 0.25 mM Ro20-1724 (a cyclic AMP-specific phosphodiesterase inhibitor), 1 mM [et32p]-ATP (3 ~tCi/reaction mixture), and 20 - 30 tzg homogenate protein in a reaction volume of 100 #1. Where indicated, either forskolin, sodium fluoride (NaF) or isoproterenol was also added, The reaction was terminated after 20 min by the addition of 800/~1 6.25% trichloroacetic acid, and a trace amount of [3H]-cyclic AMP (approximately 10,000 c.p.m.) was also added at this stage. ATP and cyclic AMP were separated chromatographically using Dowex and alumina
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columns, and the percentage fractional recovery of cyclic AMP (50 - 90%) was determined from the recovery of [3H]-cyclic AMP. Adenylate cyclase activity (pmol cyclic AMP.minLmg t protein) was derived from the 32p activity associated with the cyclic AMP fraction in the eluate from the columns which was then corrected for the fractional recovery of [3H]-cyclic AMP. l~-Adrenoceptor bindinz: I~ICYP binding to BAEC membranes was determined in triplicate samples at 25°C in the presence of 10/~M phentolamine, 5 mM MgCI:, 50 mM Tris-HCl buffer pH 7.4, selected concentrations (5 - 500 pM) of I~ICYP, and 10 - 20/~g membrane protein in a reaction volume of 200 ~d, and in the absence or presence of 50 ~tM isoproterenol to determine non-specific binding. After 2 h, membrane-bound activity was separated by negative pressure filtration through GF/C glass fiber filter paper (Whatman) using a Brandel M-24R cell harvester. Filters were washed three times with 4 ml 50 mM Tris-HCl buffer pH 7.4 with 5 mM MgC12 at 4°C, and filter-bound activity was counted on an LKB-WalIac Clinigamma 1272-004 gamma counter at 60% efficiency. Filter-bound activity was less than 10% of total radioactivity of the reaction mixtures. In preliminary experiments, binding was shown to saturate ( > 90%) within 2 h, and was linear with respect to protein within the range 10 - 50/zg. Identification of phosphorylated protein: In experiments to identify phosphorylated Gia, BAEC cultures were washed three times with phosphate-free culture medium, and were then cultured in phosphatefree culture medium in the presence of [32p]-H3PO4 0.2 mCi.m1-1 for 3 h. Either 100 nM PMA or 0.87 mM Me2SO was then added to the culture medium for the final 30 rain before harvesting. The cells were harvested, pelleted and stored as described above. Gic~ was isolated from the 32p-labeled cells by immunoprecipitation as described by Anderson and Blobel (20). Cell pellets were solubilised by boiling in 50 Izl 0.1% sodium dodecylsulfate (SDS) and then added to 200 #1 dilution buffer (1.25% Triton X-100, 190 mM NaCI, 60 mM Tris-HCl buffer pH 7.4, 6 mM EDTA). A mixture of 2 specific anti-Giot antisera (24/~1) was then added, and the mixture allowed to stand at 4°C for 18 h before centrifugation at 10,000g for 2 rain. The supernatant was added to 75 izl of a 1:1 suspension of protein A-sepharose CL-4B with continuous mixing for 2 h at 37°C. The sepharose beads were pelleted by centrifugation for 10 s in a microcentrifuge, and washed 4 times with washing buffer (0.1% Triton X-100, 0.02% SDS, 150 mM NaCI, 50 mM Tris-HCl buffer pH 7.5, 5 mM EDTA) before a final wash in the same buffer without detergent. The supernatant was removed, and 80/A sample buffer (625 mM Tris-HCl buffer pH 6.8, 10% glycerol, 1% SDS, 1% g-mercaptoethanol, 0.01% bromo-phenol blue dye) was added to the beads and boiled for 3 min. The proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and 3:p_ labeled proteins identified by gel autoradiography. Data analysis: All data are expressed as mean + S.E.M. for separate experiments. Receptor binding studies were analyzed with a non-linear iterative curve-fitting program (GrafPAD Inplot, Grafpad Software, San Diego, USA). Student's t test was used for comparisons between treatments, except where indicated when two way analysis of variance (ANOVA) was used. A p value < 0.05 was considered statistically significant.
Results Effect of PMA on adenylate cyclase activity in BAECs: BAECs were cultured in the absence or presence of 100 nM PMA for 30 min before harvesting. Cell homogenates were made, and adenylate cyclase activity was measured in the absence or presence of either 10/~M forskolin, which stimulates the adenylate cyclase catalytic subunit directly (21), or 10 mM NaF, which stimulates adenylate cyclase via the stimulatory G protein (Gs) (22), or 10/xM isoproterenol, (Fig. 1). Homogenates from cells pretreated with PMA showed significantly increased basal and stimulated adenylate cyclase activity compared with control (Fig. 1). BAECs were cultured in the absence or presence of selected concentrations (0.1 - 100 nM) of PMA for 30 min before harvesting (Fig. 2a). The enhancement of basal, 10 ~tM forskolin-, and 10 mM NaF-
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stimulated adenylate cyclase activity in cell homogenates increased with PMA concentration throughout this range (Fig. 2a). Isoproterenol (10 #M)-stimulated adenylate cyclase activity was only enhanced following exposure to the highest concentration of PMA (100 nM). BAECs were also cultured for selected times (0 - 180 min) in the presence of 100 nM PMA (Fig. 2b). The greatest enhancement of basal and stimulated adenylate cyclase activity was observed at 15 - 30 min, and had diminished towards control values by 180 min (Fig. 2 b ) . 150 ~o o
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._.R
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Fig. 1 Effect of PMA pretreatment on adenylate cyclase activity in BAEC homogenates. BAECs were cultured in the absence (open bars) or presence (hatched bars) of 100 nM PMA for 30 min before harvesting. Basal and forskolin (Forsk, 10 p.M)-, NaF (10 raM)-, or isoproterenol (Isop, 10/~M)stimulated adenylate cyclase activity was measured in BAEC homogenates. Results show the mean + S.E.M. of 14 similar experiments, ** p < 0.01 compared with untreated BAEC homogenates. ~
a 200
200
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r
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Fig. 2 Concentration and time-dependence of the effect of PMA on adenylate cyclase activity in BAECs. (a) BAECs were cultured in the presence of selected concentrations of PMA for 30 min before harvesting. (b) BAECs were cultured in the presence of 100 nM PMA for selected times before harvesting. Basal (O) and forskolin (10/~M, A)-, NaF (10 mM, II)- and isoproterenol (10 #M, * )-stimulated adenylate cyclase activity was then measured in BAEC homogenates. Each point represents the mean _+ S.E.M. of triplicate determinations and the results are a representative example of 3 similar experiments. Concentration-response relationships were determined for forskolin, NaF, and isoproterenol in homogenates from cells cultured in the absence or presence of 100 nM PMA (Fig. 3). PMA pretreatment enhanced adenylate cyclase activity at all concentrations of forskolin (0. ! - 100 p.M), NaF (1 - 100 mM), or isoproterenol (0.04 - 4 #M) (I9 < 0.0001, two way ANOVA, Fig. 3). Low concentrations of NaF (1 10 mM) activated adenylate cyclase (Fig. 3b) (22); at higher concentrations (40 - 400 mM), an inhibitory effect of NaF upon adenylate cyclase activity was observed (Fig. 3b). A similar 'bell-shaped'
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concentration-response curve with inhibition of adenylate cyclase at high concentrations of NaF ( > 10 mM) has been observed previously in cultured NG108-15 neuroblastoma cells (23), and this most probably reflects activation of the inhibitory guanine nucleotide-binding protein (Gi) as well as inhibition mediated by a Gi-independent mechanism (23).
•~ 150
100
150
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o° ~E 0 "HI , o o.1 11'01oo [Forskolin] pM
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Fig. 3 Concentration-response curves for adenylate cyclase activity in BAEC homogenates stimulated by (a) forskolin, (b) NaF and (c) isoproterenol. BAECs were cultured in the absence (©) or presence ( e ) of 100 nM PMA for 30 min before harvesting. In (c), curves were fitted according to a onesite model. Each point represents the mean + S.E.M. of triplicate determinations and the results are a representative example of 3 similar experiments. a b
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.--.
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BasalForsk. NaF Isop.
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Fig.4 Effect of staurosporine pretreatment on adenylate cyclase activity in BAEC homogenates. (a) BAECs were cultured in the absence (open bars) or presence (hatched bars) of 100nM stanrosporine for 60rain before harvesting. Basal and forskolin (Forsk, 10 #M)-, NaF (10 raM)-, or isoproterenol (Isop, 10 #M)-stimulated adenylate cyclase activity was measured in BAEC homogenates. Results are the mean + S.E.M. of 6 experiments. (b) BAECs were cultured in the absence or presence of 100 nM staurosporine for 30 min and then for a further 30 min following the addition of Me2SO or 100 nM PMA to the culture medium before harvesting. Open bars: MezSO alone; stippled bars: PMA alone; solid bars: staurosporin followed by PMA. Results are the mean _+ S.E.M. of 3 experiments, * p < 0.05 compared with BAECs pretreated with PMA alone. Effect of staurosporine: BAECs were cultured in the absence or presence of 100nM staurosporine (an inhibitor of protein kinase C) for 60 min before harvesting (Fig. 4a). Basal and forskolin (10 t~M)-, NaF (10 mM)-, and isoproterenol (10 ~tM)-stimulated adenylate cyclase activity in cell homogenates was unchanged compared with control homogenates (Fig. 4a). BAECs were cultured in the absence or presence of I00 nM staurosporine for 60 min and then in the absence or presence of 100 nM PMA for 30 min before harvesting (Fig. 4b). Basal and forskolin-, NaF-, and isoproterenol-stimulated adenylate cyclase
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activity in homogenates from cells pretreated with staurosporine and PMA was significantly lower (p < 0.05 as indicated) than that in homogenates from cells pretreated with PMA alone, but was not significantly different from that in homogenates from untreated cells (Fig. 4b). Effect of PMA on 8-adrenoceptors on BAECs: Membranes were prepared from BAECs cultured in the absence or presence of 100 nM PMA for 30 min before harvesting. Specific ~z~ICYP binding to the membranes was measured. There was no significant difference of receptor affinity or number between membranes from the PMA-pretreated cells (Kd = 53.5 + 4.8 pM; B ~ = 144.5 + 18.6 fmol.mglprotein, n = 5) and membranes from control cells (Kd = 48.8 + 7.8 pM; B~,~ = 113.9 4- 13.6 fmol.mg-lprotein, n = 5). Effect of ATP or bradykinin on adenylate cyclase activity in BAECs: BAECs were cultured in the absence or presence of 50 #M ATP (n = 5), or 10/xM bradykinin (n = 3) for 30 min before harvesting. Basal, and forskolin (10 #M)-, NaF (10 mM)-, or isoproterenol (10 #M)-stimulated adenylate cyclase activity was measured. Adenylate cyclase activity was unaltered by pretreatment with bradykinin, although basal and stimulated enzyme activity was enhanced by pretreatment with ATP (basal 21.5 + 5.7 vs 19.1 4- 5.7, p = 0.01; forskolin 79.5 4- 11.4 vs 62.4 4- 4.63, p = 0.02; fluoride 93.3 4- 17.2 vs 73.3 4- 14.2, p = 0.008; isoproterenol 57.8 ___ 8.6 vs 43.7 + 7.0 pmol cAMP.min-Lmg~protein, p = 0.04). However, the increase mediated by ATP was not inhibited by pretreatment of BAECs with 100 nM staurosporine for 60 min before harvesting. Furthermore, the effect of ATP was not mimicked by fi,^/-methylene ATP (0.1 #M - 1 raM, n = 3) nor by 2-methylthio-ATP (10 nM - 100/zM, n = 3). Effect of PMA on Gic~ phosphorylation: Gi~ was immunoprecipitated from cell homogenates which had been treated for 30 min at 37°C with 20 #g.ml -~ pertussis toxin (preactivated by heating at 37°C for 30 min in the presence of 25 mM dithiothreitol) in the presence of 100 /zCi.m1-1 [3~-p]-NAD. In pilot experiments, there was inefficient immunoprecipitation of Gic¢ with a single Gi~ antiserum. However, the addition of two anti-Gi~ antisera, with specificity for different regions of the protein, resulted in immunoprecipitation of a single protein band at 46 kDa which was radiolabeled and corresponded to [32p]_ ADP-ribosylated Gi~. This served as a positive control for the immunoprecipitation. BAECs were labeled for 3 h with [32P]-H3PO4. Thereafter, 100 nM PMA (n = 5), or 0.87 mM Me2SO alone (n = 5), was added to the cultures tbr a further 30 min. There was no evidence of phosphorylation of Gic~ under these conditions. In similar experiments, BAEC homogenates were treated for 30 min at 37°C with 100 #g.m1-1 cholera toxin (preactivated according to the protocol for pertussis toxin) in the presence of 100 p.Ci.m1-1 [32p]-NAD. Following immunoprecipitation with anti-Gsc~ antiserum, no radiolabeled protein was detected.
Discussion This study showed that pretreatment of AG04762 bovine aortic endothelial cells (BAECs) in culture with PMA, an activator of protein kinase C, led to modulation of adenylate cyclase so that basal, and forskolin-, NaF-, and isoproterenol-stimulated adenylate cyclase activity was enhanced (Fig. 1). The enhancement of adenylate cyclase activity by PMA was not seen in homogenates from BAEC cultures which had been pretreated with staurosporine, an inhibitor of protein kinase C, although staurosporine pretreatment alone had no effect on adenylate cyclase activity (Fig. 4). The effect of PMA on adenylate cyclase activity was present within 15 min of exposure and had decreased towards the untreated control values by 180 min (Fig. 2b). The rapidity of onset, and the concentration of PMA at which this effect occurred, suggest that it was mediated by phosphorylation of component(s) of the adenylate cyclase complex and not by altered rates of synthesis of these proteins. Adenylate cyclase activity was measured in cell homogenates and so the results presented here reflect true changes in activation of adenylate cyclase, and not, for example, changes in phosphodiesterase activity, which might complicate similar experiments where cyclic AMP concentration is determined in intact cells.
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Phorbol esters which activate protein kinase C modulate adenylate cyclase activity in several different cell lines (17). Depending on the cell type used, adenylate cyclase activity is increased or decreased, and phorbol ester-induced modulations of receptor sensitivity (24-26), Gs activity (27), Gi activity (28-30), and catalytic subunit phosphorylation (31) have been observed. In our study, the major effect was stimulatory leading to enhanced basal and forskolin- and NaF-stimulated adenylate cyclase activity (Fig. 3), and was presumably mediated either through Gs (increased activity), Gi (reduced activity) or the catalytic subunit of adenylate cyclase. Under the conditions of this study, Gs was present in too low a concentration to allow reproducible immunoprecipitation of Gsc~ in detectable quantities. Phosphorylation of Gsa thus remains a possible, but unlikely, mechanism by which PMA-stimulated enhancement of adenylate cyclase activity is mediated. Phosphorylation of Gsc~has never been demonstrated in intact cells, although modulation of Gso~ activity has been demonstrated in rat hepatocytes in response to protein kinase C activation (27). The mechanism of the alteration of Gs activity under these circumstances remained speculative (27). In contrast, phorbol ester-induced phosphorylation of Giot has been demonstrated in intact rat hepatocytes (29), and has been shown to be associated with reduced Gi activity and therefore increased adenylate cyclase activity. Although we were able to immunoprecipitate Gia from BAEC homogenates, there was no detectable PMA-induced phosphorylation of Gic~ under the conditions of this study. The enhancement by PMA of isoproterenol-stimulated adenylate cyclase activity in BAECs was less than that of forskolin-stimulated or NaF-stimulated adenylate cyclase activity (Fig. 1). This suggests the possibility that protein kinase C activation may have a separate and inhibitory effect on the t~-adrenoceptor. AG04762 BAECs possess f~- and ll2-adrenoceptors in approximately equal numbers, although adenylate cyclase activation is mediated predominantly via the 1~2-adrenoceptor subtype (Lefroy, unpublished observations). Others (32,33) have found that phorbol esters modulate fi-adrenoceptor function by receptor phosphorylation, and that there is a potential site for protein kinase C-mediated phosphorylation on the third cytoplasmic domain of the fJ2-adrenoceptor. Phosphorylation at this site leads to reduced coupling to Gs (33) and reduced sensitivity to epinephrine-induced adenylate cyclase activation (32). A similar mechanism could account for our observation of reduced fi-adrenoceptor function in the absence of a change in receptor density, although the PMA-dependent changes in adenylate cyclase activation by NaF and forskolin imply an additional effect of protein kinase C on either the regulatory G-proteins or the catalytic subunit. In our experiments, the effect of PMA was not mimicked by ATP or bradykinin, although activation of endothelial cell surface receptors to these agonists has been shown to stimulate phospholipase C (1,4). We observed a stimulatory effect of ATP on adenylate cyclase activity. However, both the lack of a similar effect mediated by selected ATP analogs, and also the failure of staurosporine to reverse the effect, suggested that this effect of ATP was not mediated by P2v-purinoceptors, and was independent of protein kinase C. It is possible that this effect of ATP was mediated by P2u-purinoceptors which have recently been demonstrated on BAECs (34). Alternatively, ATP may act through phosphorylation of a cell surface substrate by an extracellular (ecto-) kinase, but this mechanism is reportedly very sensitive to inhibition by staurosporine (35), and is therefore unlikely to be the explanation for our observation. Cultured BAECs have been widely used as a model system for evaluating endothelial function and after repeated passaging they maintain a stable phenotype which closely resembles that of primary endothelial cell cultures. However, there are important differences in function of endothelium derived from different sources such as large vessels compared with the microvasculature (36), and it remains to be established whether interaction between protein kinase C and adenylate cyclase activity is a feature common to all endothelial cell types. In conclusion, our data show that activation of protein kinase C in BAECs by PMA modulates adenylate cyclase activity. The mechanism for this effect may involve separate alterations in activity of the fl-adrenoceptor, G-proteins or the catalytic subunit of adenylate cyclase. Neither ATP nor bradykinin mimicked this effect under the conditions of this study, although it is possible that, under physiological conditions during which there may be more sustained activation of protein kinase C, this effect might be significant. In BAECs, the potential exists for 'crosstalk' between distinct signal transduction pathways. Interaction between these signalling pathways may be important in the control of endothelial cell function.
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Acknowledgement This research was supported in part by a P r o g r a m m e Grant from the W e l l c o m e Trust. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
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