Phorbol ester effects on α1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells

Life Sciences, Vol. 37, pp. 2389-2398 Printed in the U.S.A.

Pergamon Press

PHORBOL ESTER EFFECTS ON a.-ADRENOCEPTOR BINDING AND PP~OSPHATIDYLINOSITOL METABOLISM IN 6ULTURED VASCULAR SMOOTH MUSCLE CELLS Susanna Cotecchia, L. M. Fredrik Leeb-Lundberg, Per-Otto Hagen , Robert J. Lefkowitz and Marc G. Caron From the Howard Hughes Medical Institute Depsrtments of Medicine ~Cardiology), Biochemistry and Physiology Department of Surgery Duke University Medical Center Durham, NC 27710 (Received in final form October 7, 1985) Sum~rary Tumor promoting phorbol esters stimulate Ca ++ phospholipiddependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on ~.-adrenoeeptor binding and phosphatidylinosito] 1 metabolism In cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with blo]oglcally active phorbol esters for 15 min. to 2 hours caused a marked decresse of norep~nephrine stimulation of inositol p~9~pholipid metabolism and a 3 fold decrease in agor,ist affinity for -~I-HEAT binding to ~]-adrenoceptors in the intact smooth muscle cells. The ability of pNorbol esters to modulate ~l-adrenoceptor responsiveness suggests that activation of protein klnase C may represent an important mechanism regulating ~l-adrenergic receptor functional properties. Phorbol esters, in addition to their wide]y investigated effects ~n tumor promotion and cellular differentiation, have multiple actions in different tissues. They have been shown to modify the biological effects of several different hormones and growth factors such as insulin (1,2), EGF (3,4) and 8-adrenergic catecholamines (5,6) and their cell membrane receptors. These effects hav~+been attributed to the ability of phorbol esters to bind to and activate Ca- -phospho]ipid dependent protein kinase C (7,8). Protein kinase C is part of a cell surface signal transduction mechanism. Receptor-mediated activation of phosphatidylJnosito] (PI) metabolism generates diacylglycerol (DAG) and Inosltol trisphosphate. In turn, DAG then acts as the endogenous activator (witb other cofactors) of protein kinase C and subsequent phosphorylation ensues. Phorbol esters are thought to mimic the action of DAC due to structural similarities ~9). Inositol trispbosphate on the other hand is thought to be the signal for Ca mobilization from intracellular pools (I0). ~1-Adrenoceptors are presumably linked to this pathway through the activation of i~os~tol phospholipld metabolism in many tissues (11-16). Vascular smooth muscle cells in culture represent a convenient model system for the study of al-adrenoceptors and their coup]Ing to phosphatidylinositol turnover and calcium mobilization (15,16,17). As described above, phorbol esters have become major tools for investigating the role of protein kinase C in the regulation of different hormone-receptor

0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.

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systems. Recently, Corvera et al. (20) have reported that phorbol ester treatment of hepatocytes interfered with norepJnephrine responsiveness of these cells. In the present study we investigated the effect of phorbol esters on the functional and binding properties of ~1-adrenergic receptors of vascular smooth muscle cells in culture. Materials and Methods Materials: (-)Noreplnephrlne bitartrate, phorbo] esters and phospholipid standards were from Sigma Chemical Co.; prazosin was from Pfizer Pharmaceuticals; oxymetazoline from Draco, Lund, Sweden; HEAT (BE 2254, 2 - [ 3 - ( ~ y d r o x y phenyl)-ethyl-aminomethyl]-tetralon) was from Beiersdorf, Hamburg; ~I-HEA~was prepared by iodination of cold HEAT as3described by Engel and Hoyer (21). ~Portophosphoric acid carrier-free and [ H]myo-inositol (15.5 Ci/n~ol) were obtained from New England Nuclear Corp. Cell culture: Cultured vascular smooth muscle cells were derived from rabbit thoracic aorta. Culture conditions were as previously described (22). Growth medium was Modified Essential Medium (MEM-Gibco 410-1]00) supplemented with ]0% fetal calf serum. Experiments were performed on subcultures harvested between the fourth and eleventh passages, within one week after ~eaching confluency. Cell viability was assessed by the trypan-blue exclusion test and routlne]y exceeded 97% in both control and treated cells. Measurement of phospholipids: For phospholipid measurement cells were plated in triplicate dishes and after reaching confluency gave a consistent recovery of ~ 0 , 0 0 0 cells/dish both in contro] and treated cultures. Before phospholipid P labelling, cells were extensively washed in medium without serum and thereafter incubated in 2 ml of Krebs-Ringer-Tris buffer (117 n~ NaCI, 2.7 mM CaCI , 5.4 ~M KCI, 1.7 mM MgS0., 5 mM Tris,o 3%o BSA, pH = 37~" After lO mln equilibration in the above buffer, at 37 C, carrier freeq~ P was added at 20 ~Ci/ml concentration. After 60 min incubation at 37°C, .Zp was removed and the cells were incubated ~n fresh buffer in tbe absence and presence of (-)norepinephrine 10-~M for 15 min. In a first series of experiments, cells ~ r e treated with phorbol esters in the usual growth medium for 2 hours before P labelling. In a second series of experiments, phorbo! esters were added together with the agonlst for 15 min after - P labelling. Incubation of cells with norepinephrine was stopped by removing the buffer and adding 2 ml of ice-chilled methanol-HCl (100:1), followed by i ml of 2.4 N HCI. Samples were extracted twice under acidic conditions (23). The two combined organic phases were washed once with i ml of i0 mM KH^P04, dried under N~ and resuspcnded in 50 ~I of 200 ~i of chloroform: methanol: water ~75: 25:?). z samples were applied to i% K-oxalate impregnated silica gel 60 plastic backed plates (Merck 5506) and developed according to Jolles (24). The migration of phospbolipids was verified with authentic standards visualized with iodine vapor. Following thin layer chromatography and autorad~ography the bands were cut and counted by liquid scintillation spectrometry. ATP specific activity was determined according to Sabina et al (25) with slight modifications. For th~ determination of inositol-phosphates cells; were labelled for 72 hours with [ H]myo-inositol in the usual growth medium and then incubated Jn 2 ml Krebs-Ringer Tris buffer containing 15 n~ L!~l. After 15 min. equilibration in the above buffer at 37°C , norepinephrine 10 ~M was added for s further 15 min, in the absence or presence of phorbol esters. Phospbolipids were extracted as above and the phase extracts were collected, promptly neutralized wtih 1.5 N NaOH and diluted to 25 ml with H_0. The samples were resolved by column chromatography using i ml of Bio2Rad AGI-X8 resin (formate for~) ss described

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by Berridge et al. (26). The radioactivity distribution afte~ each elution buffer was determined and the different fractions were cellected as a single peak with 15 ml of each elution bulfer. Five m] of each eluate were counted by liquid scintillation spectrometry. Li~and Binding assay: Cells were extensively washed with ice-chilled phosphate buffered saline (PBS) and removed from the flasks using a rubberscraper. Cells were counted w3th a hemocytometer in th~ fina~ suspension. + + Binding incubation buffer consisted of PBS (without Ca , Mg , pH 7.4) and 10 ~M pargyline. Cells were incubated at a d ~ § i t y of 50,000 cells/sample (75 ~g protein) in a final volume of 0.4 ml with I-HEAl and drugs for 30 min at 25°C. Incubation was stopped by the addition of 4 ml ice-chilled PBS containing 0.1% BSA, followed by rapid filtration through Whatman GF/C filters under vacuum and four additional 5 m] washes. ~l-Agonists were dissolved in 1.6 mE sodium-ascorbate. 1251-HEAT and prazosin were dissolved in 2.5% ethanol-3 mM HCI. The fins] ~ c e n t r a t i o n of ethanol never exceeded 0.15% and that of ascorbate 0.1 mM. I-HEAT concertrations ranged from 0.025 - I nM in saturation experiments. In experiments where a single concentration of ligand was used O.] nM was chosen. Non-specific binding was determined in the presence of prazosin | ~JM, and ranged from 30-60% of total binding depending on the radioligand concentration used. Computer analysis of binding data was as described (27). Protein determination was by the method of Lowry (28). Statistical Analys~s: The statistical significance of the data sho~1 in the text was obtained by a paired two tailed student's t test. Results Figure I (control cells) shows the effect of !0 -5 M norepinephrine on the 32p labeling pattern of phospholiplds inq~ascular smooth muscle cel~s in culture. Incubation of cells prelabe]ed with -~P with norepinephrlne IO--M for 15 min caused a small but significant 57 decrease (p <0.05) of the polyphosphoinositide (PIP, PIP9) labeling accompanied by a significant increase of 24% and 58% of phesphatiflylinosit~l and phosphatidic acid respectively (p < 0.01). NorepinephrJne 10 ~M was ~he concentration which gave the maximal effect on phospholipid Iabe]ing. These effects of norepinephrine on t~e phospholipid labeling pattern were completely antagonized by prazesin 10- M (data not shown), thus indicating that they are el-adrencrgic in nature. Treatment of_~ascular smooth muscle cells with 4~ phorbol-12-myristate-13-acetate (PMA) 10 -M for 2 hours significantly decreased the effect of norepinephrine on phospholipid labeling (Fig. i). In the PMA treated cells, norepinephrlne evoked only a 9% (p
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2636 ± 489 and 1835 ±291 (n = 5, p<0.05). No change in ATP specific activity was observed after two hours of treatment with PMA'I0-JM (ATP specific activity in control and PMA-treated cells respectively 0.023 Ci/mmol and 0.029 Ci/mmo]). In a second series of experiments th~ effect o~2shorter time (15 min) of treatment of the cel~s with PMA (i0- M) after P labelling was investigated. In control cells 10 M NE (15 min) induced a significant increase of 22 _ 2% (mean + S.E.M., n = 3 p<0.05) and 43 ± 3.2%q~mean ± S.E.M., n = 3, <0.05) in phosphatidylinositol.and phosphatidic acid ~ P labelling respectlvelv. In the presence of PMA (i0 M ~ norepinephrine induced a significant increase of 9 2.7 and 8.6 ± 1.7% in ~ P labelling in phosphatidylinositol an8 phosphatidic acid respectively (mean ± S.E.M. n = 3). This response was still significantly lower than that of control cells (p<0.005). No significant change in basal ~-P radioactivity incorporation was observed in any of the pbospholipids labelled after PMA treatment with this short time of incubation. Measurement of 32p labelling of phospholipids under the conditions used above reflect at the same time hydrolysis, resynthesis and metabolism of these phospholipids. Thus, in order to examine in a more direct ~ay the effects of PMA on receptor-mediated inositol phospholipid breakdown, [ H]inositol

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z FIG. 1 Effect of PMA treatment on norepinephrine stimulation of phospholipid labeling in vascular smooth muscle cells. Phosphatidylinositol 4-monophosphate (PIP); phosphatidylinositol 4,5-bisphosphate (PIPp); phosphatid~linositol (PI); phosphatidic acid (~A). Cells were treate~ fo~o2 hours in the absence and presence of PMA 10-~M, before incubation with J~P. These data represent the norepinephrine (10- M-15 min) evoked changes in phospholipid labeling expressed as a % of the labeling observed In the absence of norepineph~Jne (basal). Results are mean ± SEM of 5 experiments performed in triplicate. In basal conditions PIP, PIP~ was 10406 ± 615 and 12533 ± 1967, PI was 2636 ± 489 ard 1835 ± 291, PA was ~2]~ ± 165 and 1245 ± 137 cpm/incubation (mean ± SEM) for control and PMA treated cells respectively. Different from control by two-tailed paired student's t test ** p<0.001, * p <0.01.

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Phorbol Effects on ~l-adrenoceptors

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phosphates generated from this breakdown were determined in control and PMAtreated cel]s after norep~nephrine stimulation in the presence of LiCI. LiCI will inhibit the formation of inositol from inositol monophosphate, thus inhibiting the resynthesis of phosphatidylinositol from these ~etabolites (13). Figure 2 shows that under these conditions norepinephrine (i0- M) stimulation for 15 min causes a significant increase of 131% in glycerol-phosphoinositol (p<0.005), 141% in inositol monophosphate (p<0.001) 128% in inositol bisphosphate (p<0.02) and 53% in inositol trisphosphate (p< 0.05). No significant accumulation of inositol was observed after norepinephrine stimulation, as expected in the_~resence of LiCI. As shown in F~gure 2 treatment of cells with PMA (10 "M) (15 min) causes a profound inhibition of norepinephrine-induced inositol phosphate formation, without any change in the basal inositol phosphate production (c.f. Fig. 2 legend).

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FIG. 2 Effect of PMA treatment on norepinephrine stimulatio~ of inositol phosphate generation in vascular smooth muscle cells. Glycerol-phosphoinositol (GPI); inositol-monophosphate (IP); Ino~itol-biphosphate (IP2) ; inositol-trisphosphate (IP3). Cells prelahelled with [5H]myo-inositol (25 ~Ci/ml) were stimulate4 for 15 mino with norepinephrine (]0- M) in the absence and presence of PM~ 10-~M in buffer containing 15 mM LiCI. Results are mean ± S.E.M. of three experiments performed ~n triplicate. In basal conditions, in control and PMA treated cells respectively. GPI was 464 ± 69 and 491 ± 69, IP was 32] ±51 and 344 ± 25, IP 2 was 193 ± 5 and 196 i 28 IP 3 was 377 ± 29 and 334 ± 29 cpm/incubation. (Mean ± S.E.M.). Different from control by two-tailed paired student's t test *** p
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As these results indicate that phorbol esters ca~ affect the ~.-adrenergic receptor mediated changes in $ ~ s i t o ! phospholipid breakdown, ~he effect of PMA treatment^was next tested on ~ I - H E A T binding to intact vascular smooth muscle cells. -2DI-HEAT binding to intact vascular smooth muscle cells reached equilibrium in 20 min at 25°C and was reversible and stereoselective. (-)Norepinephrine is I0 times more p o t e n t ] ~ a n (+)norepJnephrine in competing for the binding sites and the binding of - ~ I - H E A T is competed appropriately by various ~l-selective antagonists (data not ~ w n ) . Saturation curves analyzed by computer assisted methods revealed that ~ I - H E A T binds to a ~ingle population of sites with a K D of 140 pM and B of 60 fmo]es/lO cells (40 fmoles/mg protein). Phorbol my~istate acetat~a~reatment of vascular smooth mRscle cells for 2 hours at 10- M did not modify the affinity or B of ~=) ma I-HEAT b i n d i n g on i n t a c t v a s c u l a r s m o o t h ~ u s c l e c e l l s ( G e t a n o t s ~ o w n ) , The affinity of (-)norepinephrine (K, 0 . 8 8 X 10- M) ( F i g . 3) was d e t e r m i n e d by .1 ligand binding competition studles on the intact cells and agreed well with values previously determined on vascular smooth muscle cell membrane preparations (14). ,

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-LOG lO[(-)NORE PINEPHR INE ] (M) FIG. 3 Effect of P ~ treatment on norepinephrine competition for 1251-HEAT binding to vascular smooth muscle cells. Cells were treate~_for 2 hours in the absence and presence of PMA 10 -M. Binding was measured at -ZbI-HEAT concentrations of 0.i 0.2 nM. Data are average results from 5 different experiments~ normalized to the percentage of maximal specific binding which represented 20 fmoles/10Vcel]s. All experiments were performed in triplicate. Both competition curves were adequately modeled by a one s~te fit. EC__ values were respectively I.~9 ± 0.24 X IO--M and 3.95 ± 0.52 X IO--M for c o n t r ~ and PMA treated cells (mean ± SEM, p < 0.001) for the data shown. -

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Phorbol Effects on ~l-adrenoceptors

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Figure 3 also shows that treatment of the vascular smooth muscle cells wJthl~ ~ 10 M for two hours produced a significant decrease in the affinity of the IHEAT binding sites for norepinephr~ne. The K. values (m~an ± SEM) for (-)norepinephrine were 0.88 ± 0.16 X 10-~M and 2.6 ~ 0.5 X I0 ~M in control and PMA treated cells respectively (n=5, p < 0.01). PMA treatment also csused a two fold decrease in the affinity of the agonist oxymetazoline ±or ~l-adrenoceptors (data not shown). Figure 4 shows that PMA treatment which caused a decrease of agonist binding affinity did not change the aflinity of the ~.-adrenocep~srs for the antagonist prazosin. The K. of pra~osin in both cases w~s ~2 X 10- M. PMA tested at 1 --O concentrations up to 10 M directly in the binding assay did not affect C~l~drenoceptor binding. This is consistent with the results obtained by [ ~ I]HEAT saturation isotherms. In a second series of experiments the effect on binding of a shorter time of exposure of cells to PMA was explored. When cells were treated with PMA (10-1M) for 15 min and then PM~ was kept in the binding incubation bulfer, the same decrease of norepinephrine affinity was observed as that observed after 2 hours of PMA treatment (~ata not shown)°

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Discussion Rabbit aorta smooth muscle cells in culture represent a good experimental model for studying ~1-adrenergic receptor regulation (17,18,19). In this work we have investigated the effect of phorbol esters on vascular smooth muscle cell ~.-adrenoceptors in order to explore the potential role of protein kinase C a~tivation in regulating al-adrenoceptor activity. Norepinephrine s~imulation of a.-adrenoceptors of vascular smooth muscle cells 2 1 prelabeled with - P for one hour led to an increase in the labeling of phosphatidylinositol and phosphatid~c acid and a small decrease of polyphosphoinositide labeling. This effect is consistent with the widely reported activation of inositol lipid metabolism stimulated by ~l-adrenoceptor activation in different tissues (ii-16). Treatment of vascular smooth musc]e cells with phorbol esters for 2 hours decreased the ~1-adrenergic effect on inositol phospholipid metabolism (Fig. I). Treatmen~2of cells with phorbol esters for two hours caused a significant change of - P incorporation into phosphatidylcholine and phosphatidylinositol. This effect of the mutagen on phosphoinositides has also been recently suggested by others (8). These changes in phospholipid metabolism caused by PMA seem to be independent from its profound inhibitory effect on a.-adrenoceptor activity In fact after 15 min I " Jz exposure of the cells to PMA, no change in basal phospholipid P labelling was observed, while norepinephrine activation of phosphoinosito] metabolism was still clearly inhibited. These results are in agreement with recent findings of Corvera et al. (20) showing that phorbol esters antagonize ~.-adrenergic stimulation of hepatic glycogenolysis. Furthermore, these e~fects of phorbol esters essentially mimic those observed upon prolonged exposure of these ce]]s to norepJnephrine (Cotecchia et al. unpublished results, 18,19). In order to rule out the possibility that PMA inhibition of ~]-adrenoceptor responsiveness was due to effects other than a direct effect of PMA on inositol phospholipid breakdown we measured the norepinephrine induced ioositol phosphate formation. In fact, these metabolic parameters are a direct reflection of ~]-adrenoceptor mediated activation of phosphoinositides breakdown. Under these conditions, PMA treatment caused a profound inhibition oi Jnositol phosphate formation following al-adrenoceptors activation (Fig. 2). Recently, an inhibitory effect of phorbol esters has also been reported on muscarinic and ~1-adrenergJc receptor induced inositol monophosphate accumulation in brain slices (29). These results and our present findings suggest that protein kinase C activation might exert a negative feedback control on receptors coupled to phosphatidv]inositol turnover. In order to investigate whether the inhibitory effect of phorbol esters on ~.-adrenergic actions might be related to modification of ~.-adrenoceptor 1 binding properties, al-adrenoceptors were measured o n l ~ t a c ~ vascular smooth muscle cells. PMA did not have any direct e ~ c t on ~I-HEAT binding to intact vascular smooth muscle cells. No change in I-HEAT labeled el-receptor number and ligand affinity was found under conditions where PMA treatment decreased al-adrenergic receptor mediated effects on inositol phospholipid metabolism. However, PMA treatment caused a significant decrease of the ~l-adrenoceptor agonist affinity of vascular smooth muscle cel]s without changing the affinity for antagonists. Whether this modest decrease in affinity of the el-receptors for agonists is responsible for the pronounced inhibitory effect of phorbol esters on ~1-adrenergic metabolic activities will require further investigations. Phorbol esters have been reported to modify the affinity of different receptors for their agonists, including the EGF (3,4) and insulin receptors (1,2). The decrease in aflinity of EGF and insulin receptors caused by phorbol esters is associated with receptor phosphorylation (3],32) suggesting that these receptor proteins may be substrates for protein kinase C.

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Thus, it seems possible that the effect of phorbol esters on the agonist affinity of ~1-receptors and the uncoupling of their ability to promote inositol phospholipid turnover might be mediated by phospholylation of the ~I receptor. If this occurs it would he highly analogous to the case of the adenylate cyclase coupled ~-adrenergic receptors which are also uncoupled by phorbol ester treatment (5,6), which leads to receptor phosphorylation. Acknowledgement We thank Donna Addison for expert secretarial Cotecchia is supported by a fellowship from FORMEZ Center of the Southern Italian Development Agency) Mario Negri Institute, Milan, Italy. Dr. Hagen is N.I.H. #HL15448.

assistance. Dr. Susanna (Professional Education directly ~dministered by supported by a grant from the

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A. HANCOCK, A. DE LEAN and R. J. LEFKOWITZ, Mol. Pharnmcol. 16, i-9 (1979). O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, J. Biol. Chem. 193, 265-275 (1951). M. V., TAYLOR, 3. C. METCALFE, T. R. HESKETH, G. A. SMITH AND 3. P. MOORE, Nature 312, 462-465 (1984). R. LABARCA, A. JANOWSKY, T. PATEL AND S. M. PAUL, Biochem. Biophys. Res. Commun. 123, 703-709 (]984). S. JACOBS, N. E. SAHYOt~, A. R. SALTIEL and P. CUATRECASAS, Proc. Natl. Acad. 8ci. USA 80, 6211-6213 (1983). P. J. DAVIS and M. P. Czech, J. Biol. Chem. 259, 8565-8549 (1984).