Sustained Diacylglycerol Formation from Inositol ... - Semantic Scholar

Val. 261,No.13, Issue of May 5, pp. 59013906,1986 Printed in U.S.A.

THE JOURNALOF BIOLOGICAL CHEMISTRK 0 1986 by The American Society of Biological Chemists, Inc.

Sustained Diacylglycerol Formation from InositolPhospholipids in Angiotensin 11-stimulated Vascular Smooth Muscle Cells* (Received for publication, July 8, 1985)

Kathy K. GriendlingSB, Susan E. Rittenhouse$ll((,Tommy A. Brock**$$, Laurie S . EksteinSg, Michael A. Gimbrone, Jr.**$$,and R. Wayne Alexander80 From the Departments of $Medicine and **Pathology, Haruard Medical Schooland the §Cardiovascular,TiHematoology, and Massachusetts 02115 $$Vascular Research Diuisiow, Brighan and Women% Hospital, Boston,

Angiotensin I1 acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin I1 induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin I1 (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15s (227 +. 19%control) and at 5 min (303 f 23%control). Simultaneous analysis of labeled inositol phospholipids shows that a t 15 s phosphatidylinositol 4,5-bisphosphate (PIPz) and phosphatidylinositol 4-phosphate (PIP) decline to 52 f 6% control and 63 k 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIPz and PIP have returned toward control levels (92 f 2 and 82 f 4% control, respectively), while PI has decreased substantially (81 +. 2% control). The calcium ionophore ionomycin (15 MM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4p-Phorbol12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin I1 induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinaseC-mediatedinhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin 11 inositol 1,4,5trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.

Polyphosphoinositidemetabolism recently has been shown to be important in the generation of intracellular signals for hormones which act by increasing cytosolic free calcium(reviewed in Refs. 1-4). Attention has been focusedon the early

breakdown of PIP2,1the resultant formation of IP3, and the release of calcium fromnon-mitochondrial intracellular storage sites (5). The other product of PIP2 hydrolysis, diacylglycerol (DG), has been proposed as a second messenger for activation of a calcium- and phospholipid-sensitive protein kinase (protein kinase C) (6),but relatively few systematic investigationsof hormonally inducedDG formation have been reported. Rasmussen and Barrett (7) havesuggested that each of these two messengers may have a unique temporal role: the IPa/calcium system being responsible for an initial transient cellular response and the DG/protein kinase C system being responsible for a sustained phase of the cellular response to various hormonal stimuli. There is at present, however, some question as to whether DG formation is sustained. In platelets, production of DG in response to thrombin is transient, and DG levels return to control values within 2 min (8). In contrast, theangiotensin 11-inducedincrease in DG formation in adrenal glomerulosa cells is sustained for at least 10 min (9). In hepatocytes, two studies report a sustained increase in DG in response to vasopressin (10,11),while another reports only a transient increase (12). While IP3presumably can be formed only from PIP, breakdown, DG could alsobe formed fromphospholipase C-mediated hydrolysis of PIP or PI. AlthoughBerridge (3) has suggested that phospholipase C acts on PIP,, rather than on PI, to generate DG and IPS in equimolar amounts, in angiotensin 11-stimulatedadrenal glomerulosa cells,PIP and PIP2 are returning to control levels at a time when DG levels are still increased (9). Many other cell types which exhibit sustained physiological responses to stimulation bycalciummobilizing hormones also display only transient breakdown of the polyphosphoinositides(13-15). Wilson et al. (16) have demonstrated that purified phospholipase C can directly hydrolyze PI, PIP, and PIPz in unilamellar vesicles at a rate dependent upon ambient calcium concentration and phospholipid composition of the membrane. In intact cells, although a delayed agonist-stimulated PI turnover has been observed for many years (2), decreases in PI are usually attributed to phosphorylation by kinases rather than to hydrolysis by phospholipase C (5). Wilson’s observation in vesicles raises the

’

The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; IP,, inositol trisphosphate; DG, diacylglycerol;PIP, phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; VSMC, vas* This work was supported by National Institutes of Health Grants cular smooth muscle cells; NEPES, 4-(2-hydroxyethyl)-l-piperazineHL 20054/HL 35013, HL 22602, HL 29763, HL 35797, and HL 07042. ethanesulfonic acid; PMA, 48-phorbol 12-myristate 13-acetate; 4-0The costs of publication of this article were defrayed in part by the methyl PMA, 4P-phorbol 12-myristate 13-acetate 4-0-methyl ether; payment of page charges. This articlemusttherefore be hereby PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylmarked “advertisement” in accordance with 18 U.S.C. Section 1734 ethanolamine; PA, phosphatidic acid; IP, inositol phosphate; IP2, solely to indicate this fact. inositol bisphosphate; [Ca2+Ii, ionized cytosolic calcium; EGTA, (1 Established Investigator of the American Heart Association. [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

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Sustained Diacylglycerol Formation in Vascular Smooth Muscle

possibility that PI could be the source of agonist-stimulated DG production in intact cells after polyphosphoinositide breakdown has decreased. There arefew studies inwhich the time courses of agonistinduced DG and IP, production and of PI, PIP, and PIP, hydrolysis have been characterized. The objectives of the experiments reported here were to define in cultured vascular smooth muscle the pattern ofDG and IP, formation after angiotensin I1 stimulation, to establish the source of the DG formed, and to gain insight into the mechanisms controlling phospholipase C-mediated hydrolysis of the various potential phosphoinositide substrates. EXPERIMENTAL PROCEDURES

Cell Culture-VSMC were isolated from rat thoracic aorta by enzymatic dissociation as described previously (17, 18). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, glutamine, and antibiotics and were passaged twice a week by harvesting with trypsin-versene and seeding a t a 1:4 ratio in 75-cmZflasks. For experiments, cells between passage levels 4-25 were seeded into 35- or 100-mm dishes (2 X lo4 cells/cm2), fed every other day, and used a t confluence (2-6 days). Phospholipid Labeling and Extraction-VSMC cultures were incubated with either [3H]myoinositol (25 pCi/ml) for 48 h in standard growth medium or [3H]arachidonic acid (1pCi/ml) for 3 h in serumfree Dulbecco’s modified Eagle’s medium containing 0.2 mg/ml bovine serum albumin. Unincorporated isotope was removedby washing cultures ina warm balanced salt solution of the following composition (mM): 130 NaCl, 5 KCl, 1 MgC12, 1.5 CaC12, 20 HEPES (buffered to pH 7.4 with Tris base). Cells were then incubated for 20 min in 2 ml of balanced salt solution a t 37 “C. In some experiments, cells were incubated for an additional 5 min in 100 nM PMA and then exposed to angiotensin I1 in the continued presence of PMA; otherwise, the balanced salt solution was replaced by 1ml of fresh buffer containing angiotensin 11. Unless otherwise indicated, a maximal concentration of angiotensin I1 (100 nM) was used as a stimulus. The reaction was terminated after various intervals by rapid aspiration of the buffer and addition of 1 ml of chloroform/methanol/HCl (1:2:0.05) for phospholipid extraction or chloroform/methanol (1:2) for neutral lipid extraction. Organic and aqueous phases of the extract plus a 0.5-ml rinse were separated by addition of 400 pl of chloroform and 400 plof distilled water, followed by centrifugation. The upper phase was washed twice with 800 pl of chloroform, and the chloroform phases were pooled and concentrated under nitrogen. PI, PIP, PIP2, PA, PS, PC, and PE were resolved by thin layer chromatography as described previously (19). For neutral lipid (DG, triglyceride) determinations, lipids were dissolved in 150 pl of chloroform and applied with appropriate standards to preheated 250-pm Silica G plates (Uniplate, Analtech). Plates were placed in equilibrated tanks containing benzene:diethyl ether:ammonia (100:800.2, v/v). Separation of the neutrallipids was verified using a two-dimensional chromatography system. Samples were applied to heat-activated Silica Gel 60 plates and developed in the first direction in the solvent system diethyl ether:hexane:acetic acid (70301, v/v). The plates were then air dried for 1h and developed in the second direction, in the solvent system benzenediethy1 ether:ethanol:ammonia (100840.2, v/v). All resolved lipids were visualized by iodine staining and, aftertreatment with EN3HANCE spray (New England Nuclear), by autoradiography on Kodak X-OMAT AR film. Upper phase extracts containing [3H] myoinositol-labeled inositol phosphates were concentrated under nitrogen and resolved by the column chromatography method of Downes and Michell(20) as described previously (21). Alllipids were quantified by liquid scintillation spectrophotometry. Measurement of Cell Calcium-Fura-2, a calcium-sensitive dye, was used to monitor changes in [Ca2+]i,using procedures described previously for quin-2 in these cells (18).Cell suspensions (5 X lo6 cells/ ml) were prepared using 8 X 100-mm cultures, incubated for 30 min with 5 p~ fura-2/acetoxymethyl ester, washed twice (200 X g, 3 min) with BSS, and resuspended to 4 X lo6 cells/ml with BSS containing 10 mM glucose. The cells were kept at room temperature, centrifuged in a Beckman Microfuge (model B) prior to use, and resuspended to 2 X IO6 cells/ml. Measurements of fluorescence were made a t 37 “C using a Spex fluorolog-2 spectrofluorimeter (excitation 340 nm, slit 2.25 nm; emission 505 nm, slit 4.5 nm) equipped with a thermostated cuvette holder, stirring apparatus,and chartrecorder. The calibration

of fura-2 fluorescence and calculation of [Ca2+Iiwere determined using digitonin (50 p ~ and ) Tris/EGTA (6 mM, pH 10) to obtain maximal (F& and minimal (Fmb)fluorescence signals, respectively. [Ca2+liwas calculated using the formula: [Caz+li= Kd ( F Fmin)/ (Fma - F ) where F was the fluorescence intensity of fura-2 within the cell and Kd was 224 nM (22). Materials-The supplies and vendors used in this study were as follows: [3H]myoinositol (15 Ci/mmol), Amersham, Chicago, IL; [5,6,8,9,11,12,14,15-3H]arachidonic acid (83.8 Ci/mmol), New England Nuclear; Dulbecco’s modified Eagle’s medium, calf serum, glutamine, penicillin and streptomycin, MA Bioproducts, Walkersville, MD; angiotensin 11, Peninsula Laboratories, Belmont, CA, bovine serum albumin (Pentex), Miles Laboratories, Elkhardt, I N lipid standards (phosphatidylinositol, phosphatidic acid, phosphtidylserine), Avanti Polar Lipids, Inc., Birmingham, AL; 1,2-diolein,Serdary Research Labs, Ontario, Canada; phorbol esters (PMA, 4-0-methylPMA), Sigma; fura-2/acetoxymethyl ester, Molecular Probes, Inc., Junction City, OR.

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RESULTS

Time Course and Dose Dependence of Diglyceride Formation-Angiotensin 11(100 nM) stimulation of rat aortic VSMC cultures induced a substantial increase in DGwhichwas detectable at 5 s and was sustained for at least 20 min (Fig. 1). DG formation consistently showed two distinct phases. An early peak occurred at 15 s (227 +- 19% control, mean +S.E., n = 16), while a larger late peak occurred at 5 min (303 k 23% control, n = 17). Both phases were dose dependent, with the threshold for angiotensin 11-induced DG formation occurring at 1nM angiotensin I1 and maximal DG production occurring at 100 nM angiotensin I1 (Fig. 2). This observation agrees well with the dose dependence of angiotensin II-induced IP, formation in these cells (21). Phosphoinositide Hydrolysis and Inositol Phosphate Formation-To determine the source(s) of the observed DG, angiotensin 11-induced DG formation and hydrolysis of PI, PIP, and PIP, were simultaneously measured at both 15 s and 5 min (Fig. 3). Cells labeled with either [3H]myoinositol or [3H]arachidonic acid showed essentially the same results. At the early time point, PIP and PIPz decreased to 63 +- 5 and 52 f 6% control, respectively (n = 7, p < 0.001), while there was little change in the level of PI (96 f 2% control, n = 15,p > 0.05). By 5 min, however, PIP and PIPzhad returned toward, but had not reached, control levels (82 f 4 and 92 f 2% control, respectively, n = 9, 10, p < 0.011, while PI had decreased substantially (81 f 2% control, n = 18,p 0.001). There was no significant hydrolysis of the other major phospholipids after 5 min of angiotensin I1 stimulation (PC and PE: 100 & 1%control (n = 6, p > 0.1); PS: 94 f 3% control

I

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5

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t

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Time ( m i d

FIG. 1. Time course of angiotensin 11-stimulateddiacylglycerol,formation. Cultured VSMC were prelabeled with t3H]arachidonic acid (1pCi/ml) for 3 h and thenexposed to angiotensin I1 (100 nM) for various periods. This time course is representative of three experiments which yielded similar results.

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88

Controt Ang II (100 nM)

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ANGIOTENSIN (-log M)

FIG. 2. Dose dependence of angiotensin 11-induced diacylglycerol formation. Cultured VSMCwere prelabeled with [‘€I] arachidonic acid (1&i/ml) for 3 h, and thenexposed to angiotensin I1 for 5 min (e-”.) or 15 s ( O d ) .Each cume is representative of three experiments which yielded similar results.

(n = 6, p > 0.1)) nor was there any change in triglyceride levels (107 f 5% control, n = 14, p > 0.1). Lysophosphatidylinositol had decreased 5 min after angiotensin 11 stimulation (82 & 2% control, n = 5, p < 0.001), indicating an absence of PI-specific phospholipaseAPactivity. As shown in Fig. 3B,the radioactivity lost from labeledPIP and PIPz at 5 rnin appears to be insufficient to account €or the large increasein DG, especially sincethere was a concomitant sustained increase in PA, a major metabolite of DG (8,154 +. 963 cpm (control) uersw 14,948 f 1,497 cpm (angiotensin 11),p < 0.02). The large drop in PI at5 min suggests that PIhydrolysis may contribute to sustainedDG formation. In support of this hypothesis, IP, the other product of phospholipase C action on PI, remained elevated in these cells at times (2-20 min) at which only minor increases in IP2 and IPSwere observed (Fig.4B). As noted above, there was a small continued hydrolysis of PIP and PIP2 in the presence of angiotensin I1 (Fig. 44).This observation was confirmed by the persistence of a modest elevation in IPZ (141 rk 37% increase, D = 4). and IP3 (42 21% increase, n = 3) at 5 min (Fig. 4B). At the early time point (15 s), 95% of the total IP, formed by these cells was the physiologically active 1,4,5-IP3isomer. By 10 min, fully 77% of the total 1P3formed was 1,4,5-1P3as measured by high pressure liquid chromatography.2 Effect of Calcium on Phosphoinositide Hydrolysis and DG Formation-Because of the observation that calcium stimulated PI hydrolysis in an isolated enzyme system (16), the effect of calcium on phospholipase C-mediated hydrolysisof the phosphoinositides was investigated in intact cells in an attempt to determine whether calcium plays a pivotal role in altering the substrate for phospholipase C. The calcium ionophore ionomycin (free acid, 15 PM) caused an immediate sustained increase in [CaZ*li at least as large as that seen transiently with LOO nM angiotensin 11 (Fig. 5). At this concentration of ionomycin, cells remained viable, as judged by their ability to exclude trypan blue. At 15 s, the ionomycininduced increase in (Ca2+]iwas not associated with changes in DG formation or phosphoinositide hydrolysis. However, after 5 min of sustained elevation of [Ca2+Ii,there was an increase above control in DG (1,987 f 161 ( n = 7) to 5,209 4 707 (n= 8) cprn, p 4 0.01), PA (3,221 4 358 to 4,407 -+ 402 cpm, n = 7, p < 0.05), and PIP (2,921 r+, 384 (n = 6) to 4,996

*

Dr. C. Peter Downes, unpublished observation.

DG PIP, PIP P1 FIG.3. Effect of angiotensin II on phusphuinusitide hydrolysis and diacylglycerol formation at A, 15 s and B, 5 min. Cultured VSMC were prelabeled with [3H]arachidonic acid (1 pCi/ ml) for 3 h and then exposed to angiotensin 11 (100 nM) for the periods indicated. Each bur represents the mean IS.E. of duplicate determinations from at least 3 experiments.

k 413 (n = 5) cpm, p < 0.01). Changes inother phospholipids at 5 min were not significant: PI (126,622 +- 6,917 to 114,121 & 7,219 cpm, n = 6, p > O.l), PIPz (3,564 +- 336 to 3,288 f 321 cpm, n = 5, p > O.l), PS (20,139 +- 2,408 (n = 6) to 18,726 & 2,003 (n = 7 ) cpm, p > O.l), and PC and PE (220,460 18,337 (n = 6) to 210,312 2 11,159 (n = 7) cpm, p > 0.1). Although angiotensin 11-induced increases in [Ca2++Ii return to basal levels by 5 min, there appears to be a persistent increase in calciumpermeability in cultured VSMC? (23) which is presumably accompaniedby an increase in calcium efflux. T o test thepossibility that a high intracellular calcium environment in the region of the cell membrane could contribute to the breakdown of PI, we abolished sustained hormonally induced calcium influx by preincubating cells with EGTA (2 mM,1min). Removing extracellular calcium had no effect on PI hydrolysis at 5 min (angiotensin 11, 85 -C 5%

K. K. Griendling, S. E. Rittenhouse, T. A. Brock, L. S. Ekstein, M. A. Gimbrone, Jr., and R. W. Alexander, unpublished observation.


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FIG. 5. Effect of angiotensin I1 and ionomycin on [CaS'li. Cultured VSMCwere incubated with fura-2/acetoxymethyl ester, washed, and resuspended to a final concentration of 2 X IO6 cells/ml. Measurements of [Ca'+Jiwere made spectrofluorimetrically. Top, typical fura-2 fluorescence in response to 100 nM angiotensin I1 (peak: 6.1 +- 0.5-fold increase, n = 9). Bottom, typical fura-2 fluorescence in response to 15 ,AM ionomycin. Addition of each agent is indicated by the arrow. Calculated values for [CaZ+liare indicated at theright.

lB 2!

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TABLEI Effect of PMA on angiotensin 11-stimulated diglyceride formation Cultured VSMC were labeled with [3H]arachidonicacid (1 pCi/ml) for 3 h and thenwere either exposed to angiotensin I1 (100 nm, 15 s) or preincubated with PMA (100 nM, 5 min) and exposed to angiotensin II in the continued presence of PMA. Values are expressed as mean -r- S.E. of duplicate determinations from at least 4 experiments. * indicates significance ( p < 0.05). DG (15 s)

Condition

DG (5 min)

% increase

TIME (mid

FIG. 4. Time course of angiotensin 11-stimulated phospholipid breakdown and inositoI phosphate production. Cultured VSMC were prelabeled with [3H]myoinositol (25 pCi/ml) for 48 h and then exposed to angiotensin I1 (100 nM) for different intervals. A , phospholipid hydrolysis. Data are expressed as per cent control. A-A, PI, control = 351,300 cpm; o"--o, PIP, control = 10,423 cpm; ."3, PIP, control = 11,757 cprn. B , inositol phosphate production. Data are expressed in cpm. A-A, IP; o"--o, IP,; @"4, IPS.Each point represents the mean of duplicate determinations. Two additional experiments with fewer time points yielded similar results.

Angiotensin I1

117 +- 14

*

164 -1- 28

Angiotensin I1 + PMA

72 f 10

233 +- 42

94 k 6% control, n = 5, p < 0.01) but had no effect on PI hydrolysis (angiotensin 11, 98 k 3% control; angiotensin I1 plus PMA, 98 It- 4%control, n = 7 , p > 0.1). In contrast, PMA did not inhibit DG formation at 5 min (Table I). The less potent phorbol ester, 4-0-methyl PMA, had no effect on angiotensin 11-induced DGformation. Thus, PMA appears to inhibit selectively the early, but not the late phase, of DG production. DISCUSSION

The results of the present study demonstrate that angiotensin 11-induced DGformation is sustained in cultured rat aortic control; angiotensin I1 plus EGTA, 82 f 4% control, n = 3, p VSMC, irrespective of inhibition of early changes in DG and IP, by phorbol ester.The existence o f two peaksin DG > 0.1). Effect of PMA on Phosphoinositide Hydrolysis and DG production and the difference in the profiles of phosphoinoFormation-We have previously shown that PMA, an exoge- sitide hydrolysis at early (15 s) and late ( 5 min) times suggest nous activator of protein kinase C, inhibited angiotensin II- a shift in the substrate for phospholipase C from the polystimulated polyphosphoinositide hydrolysis and calcium flux phosphoinositides to PI with time. Angiotensin 11 induces in these cells (24). To test whether PMA would also inhibit rapid breakdown of PIPz and PIP and delayed hydrolysis of angiotensin 11-induced breakdown of PI, we investigated the PI which is not detectable until after 2 min of hormonal effect of PMA on angiotensin 11-stimulated hydrolysis of all stimulation and does not appear to require a sustained elethree phosphoinositides and on DG formation. Basal levels of vation of calcium. PIP, and PIP breakdown are attenuated all the phospholipids were unchanged by a brief incubation after 2 min while PI hydrolysis is not, suggesting a selective with PMA (100 nM,5 min). However, early (15 s) angiotensin inhibition of polyphosphoinositide hydrolysis during the sus11-stimulated DG formation was inhibited by PMA (43k 9% tained cellular response. Although many other studiesusing different cell types have inhibition, n = 4, p < 0.02, Table I), which is consistent with the effect of PMA on angiotensin 11-induced IPSformation demonstrated rapid hydrolysis of the polyphosphoinositides and cytosolic calcium changes at 15 s (24). In agreement with (13, 14, 25) and late turnover of PI (14, 261, there are few our previous observations (24), PMA also attenuated early studies whichhave measured concurrent changes in DG. breakdown of PIP (angiotensin II,79 k 7% control; angioten- Sustained DG formation (10 min) in vasopressin-stimulated sin I1 plus PMA, 103 f 6% control, n = 5, p < 0.05) and PIPz hepatocytes was not accompanied by hydrolysis of PIP and (angiotensin 11, 65 k 4% control; angiotensin I1 plus PMA, PIPz (10). However, the authors did not examine late PI

Sustained Diacylglycerol Formation in breakdown and attributed thelate DG formation to agoniststimulated lipogenesis. In adrenal glomerulosa cells stimulated with angiotensin 11,DG formation was biphasic and was sustained for a t least 10 min, but no data relevant to determining its source were reported (9). In cultured VSMC stimulated with angiotensin 11, DG formation previously has not been measured directly. However, Smith et al. (27) found that PA, the product of DG kinase action on DG, was increased 1.5-fold by100 nM angiotensin I1 at 5 min, which is consistent with our observations. In addition, Nabika et al. (28) reported a sustained increase in IP formationafter angiotensin I1 stimulation of VSMC. They also demonstrated an increase in total phosphoinositide hydrolysis over 20 min but did not distinguish among PI, PIP, and PIP2. Stimulated loss of PI in other systems has been attributed to itsphosphorylation by kinases in theplasma membrane to replace the polyphosphoinositides hydrolyzed by phospholipase C (3). Although some PI is shuttled through this pathway, replenishment of the PIP and PIP2pools could account for only 7% of the PI lost in VSMC. In order for PI to be consumed solely by kinase activity, PIP2 would have to be hydrolyzed and replaced with a much greater rate of turnover than was observed in the initial response. Hydrolysis of IP3 and IP2would have to accelerate as well, since there was no progressive accumulation of IP2or IP3. Although we cannot rule out thepossibility that increased turnover could account for the late PI hydrolysis, the appearance of a large sustained increase in both IP and DG is consistent with a direct action of phospholipase C on PI. Direct hormonally induced phosphodiesteratic cleavage of PI has recently been demonstrated in mouse pancreatic lobules (29). The mechanism underlying the delay in induction of PI hydrolysis is at present unknown but might be related to a competition among phosphoinositide substrates for phospholipase C. Wilson et al. (16) showed that in unilamellar phospholipid vesicles of a phosphoinositide composition similar to that found in the cell membrane, PI and the polyphosphoinositides competed for a limiting amountof phospholipase C. It is thus possible that PI is hydrolyzed only after levels of PIP and PIP2 have decreased. It seems unlikely, however, that changes in substrate concentration could alone account for this late PI breakdown in VSMC, since PI continues to be hydrolyzed even when polyphosphoinositide levels are returning toward base line (Fig. 4A) and when hydrolysis of polyphosphoinositides is blocked by exposure of VSMC to PMA. Other possible explanations for delayed PI hydrolysis include the existence of two distinct phospholipase Cs (16) with temporal differences in activation, a shift in phospholipase C activity due to agonist-induced changes in pH (30), or the existence of different domains within the cell selectively enriched in polyphosphoinositides or PI (2) and sequentially activated by the receptor-angiotensin I1 complex. Alternatively, the lateonset of PI hydrolysis might be related to theangiotensin II-induced increase in [Ca2+Ii,since PI breakdown by purified phospholipase C is calcium dependent (16). However, in cultured VSMC, an increase in [Ca2'li does not appear to be a sufficient explanation for the late angiotensin II-induced activation of direct PI hydrolysis by phospholipase C. PI hydrolysis was not observed during the time when [Ca2+]iwas elevated but became detectable only after the calcium levels had returned to base line (2 min, Fig. 5 ) . Furthermore, abolishing putative high membrane calcium flux during the tonic phaseof stimulation ( 5 min) by removing extracellular calcium with EGTA did not attenuate the late PI breakdown. In addition, rapidly increasing cytosolic calcium with ionomycin (15 s) did not increase PI hydrolysis. A

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5-min incubation with calcium ionophore causing a sustained increase in [Ca2+]i,however, did cause accumulation of both DG and PA, although to a lesser extent than did angiotensin 11 (combined DG plus PA increase: ionomycin, 238%; angiotensin 11, 338%). The source of ionophore-stimulated DG is unclear, since ionomycin induced small, but not statistically significant, changes in each of the major phospholipids. Bocckino et al. (11) have shown by fatty acid analysis that calcium ionophore-induced DG formation in hepatocytes derives from multiple sources, and such may be the case in our study, Despite the ability of calcium to stimulate DG formation, it does not reproduce the late phase of the hormonal response, since the pattern of phosphoinositide metabolism induced by angiotensin I1 is markedly different from that seen with calcium ionophore alone. Specifically, angiotensin I1 induced a highly significant 19%breakdown in PI and an 18% breakdown in PIP at 5 min, while ionophore caused an insignificant breakdown in PI and a 70% increase in PIP. Thus, while we cannot exclude a role for calcium in initiating events leading to theangiotensin II-induced hydrolysis of PI, the late phase of the hormonal response does not appear to require a sustained elevation of cytosolic calcium. The mechanism for the shift in phospholipase C activity away from the polyphosphoinositides is most likely related to generation of DG itself. The ability of phorbol ester to inhibit the early but not thesustained DG formation is suggestive of a role for DG-activated protein kinase C in the selective inhibition of phospholipase C-mediated breakdown of PIP2 and PIP. In support of this hypothesis, we have previously demonstrated that PMA and l-oleoyl-2-acetylglycerolinhibit angiotensin II-stimulated IP2 and IP3 formation at 15 s in these cells (24). The observation that phospholipase C directly hydrolyzes all the phosphoinositides in intactVSMC followingangiotensin I1 stimulation has several implications. The existence of multiple sources for DG provides a mechanism for selectively maintaining DG formation, while permitting a transient peak in 1,4,5-IP3 formation. Maintenance of increased DG levels may ensure that those cellular responses which are controlled by protein kinase C can be sustained throughout agonist stimulation. Transient large changes in IP3 resulting from attenuation of early PIP2 hydrolysis suggest a mechanism for the observed transience of the increase in [Ca2+Ii(21) and of the calcium-calmodulin-dependent phosphorylation of the myosin light chain (31). The small persistent elevation in 1,4,5-IP3 may function to inhibit refilling of intracellular storage sites during tonic agonist stimulation. In support of this hypothesis, we have observed that total cell calcium remains depressed for upto 2 h in the presence of angiotensin 11.3The physiological significance of the residual IP2 remains to be determined. Although phosphoinositide hydrolysis by phospholipase C would correlate well with the phospholipid changes measured here, there are other possible explanations for the observed decrease in PI and thelate increase in DG. If isotopic equilibrium is not reached before the agonist is added, the measured changes can be partially due to changes in specific activity of relevant phospholipid pools. However,equivalent results were obtained for those lipids which were labeled with either [3H] arachidonic acid or [3H]myoinositol(PI, PIP,PIP2),and each isotope labeled these three constituents in the same proportion(942.63.4). Therefore, changes in radioactivity most likely reflect actual changes in mass. PI, however, could also be metabolized by phospholipase A2. This enzyme does not appear to be activated by angiotensin I1 in VSMC, since we observed a small drop, rather than a rise, in lysophosphati-

5906


dylinositol. Thereare also other possible sources for DG, particularly other phospholipids (PC, PE, PS) 01 triglyceride.

(1983) J. Biol. Chem. 258,5716-5725 11. Bocckino, s. Bv Blackmore, F-, and Exton, J- H. (1985) J. Bwl. Chem. 2 6 0 , 14201-14207 *lthough we found no difference in any Of these parameters 12. Hughes, B. P., Rye, K. A., Pickford, L. B., Barritt, G. J., and in cells stimulated with angiotensin I1 for 5 min, we cannot Chalmers, A. H. (1984) Biochem. J. 222,535-540 rule out thepossibility that small changes, Particularly in the 13. Creba, J. A., Downes, C. P., Hawkins, P. T., Brewster, G., Michell, phospholipids, might contribute to the observed increase in R. H., and Kirk, C. J. (1983) Biochem. J. 212,733-747 14. Rebecchi, M. J.,and Gersheneorn. M. C. (1983) Biochem. J. 2 1 6 , DG. -. 287-294 . In summary, we have shown that angiotensin 11-induced 15. Litosch, I., Wallis, C., and Fain, J. N. (1985) J. Bwl. Chem. 2 6 0 , DG formation in cultured VSMC is sustained for as long as Fi464-5471 "_. 20 min after initialexposure to theagonist. Our data strongly 16. Wilson, D. B., Bross, T. E., Hofmann, S. L., and Majerus, P. W. suggest a time-dependent variation in the rate of hydrolysis (1984) J. Biol. Chem. 259,11718-11724 of all three phosphoinositides by phospholipase C. Initially, 17. Gunther, S., Alexander, R. W., Atkinson, W. J., and Gimbrone, M. A., Jr. (1982) J. Cell Bwl. 92, 289-298 angiotensin I1 stimulates rapid hydrolysis of PIP and PIP2. T. A., Alexander, R. W., Ekstein, L. S., Atkinson, W. J., The &glyceride produced, probably through activation of 18. Brock, and Gimbrone, M.A., Jr. (1985) Hypertension 7,Suppl. I, Iprotein kinase C , appears to feed back to partially attenuate 105-1-109 further hydrolysis of the polyphosphoinositides. The larger 19. Rittenhouse, S. E. (1983) Proc. Nutl. Acud. Sci. U. S. A. 80,54175420 pool of PI then becomes a major source for continued DG production. The significance of multiple substrates for phos- 20. Downes, C. P., and Michell, R. H. (1981) Biochem. J. 1 9 8 , 133140 pholipase C is that DG formation is not necessarily coupled R. W., Brock, T. A., Gimbrone, M.A., Jr., and to IP, generation, suggesting a possible role for DG in the 21. Alexander, Rittenhouse, S. E. (1985) Hypertension 7,447-451 mediation of the sustained VSMC response to angiotensin 11. 22. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Bwl. Chem. 260,3440-3450 Acknowledgments-We wish to thankDr. C. Peter Downes, Smith, 23. Capponi, A. M., Lew, P. D., and Vallotton, M. B. (1985) J. Biol. Chem. 260, 7836-7842 Kline and French Research Ltd., United Kingdom, for analyzing the angiotensin 11-stimulated IP3 on his high pressure liquid chromatog- 24. Brock, T. A., Rittenhouse, S. E., Powers, C. W., Ekstein, L. E., Gimbrone, M. A., Jr., and Alexander, R.W. (1985) J. Biol. raphy system. Chem. 260,14158-14162 25. Agranoff, B. W., Murthy, P., and Seguin, E. B. (1983) J. Bwl. REFERENCES Chem. 258,2076-2078 1. Michell, R. H. (1975) Biochim. Biophys. Actu415.81-147 26. Troyer, D. A., Kreisberg, J. I., Schwertz, D. W., and Venkatach2. Hokin, L. E. (1985) Annu. Rev. Biochem. 5 4 , 205-235 alam, M.A. (1985) Am. J. Physwl. 2 4 9 , F139-Fl47 3. Berridge, M. J. (1984) Biochem. J. 220,345-360 27. Smith, J. B., Smith, L.,Brown, E. R., Barnes, D., Sabir, M. A., 4. Williamson, J. R., Cooper, R. H., Joseph, S. K., and Thomas, A. Davis, J. S., and Farese, R. V. (1984) Proc. Natl. Acud. Sei. P. (1985) Am. J. Physiol. 2 4 8 , C203-C216 U. S. A. 81,7812-7816 5. Berridge, M. J., and Imine, R. F. (1984) Nature 312,315-321 28. Nabika, T., Velletri, P. A., Lovenberg, W., and Beaven, M.A. 6. Nishizuka, Y. (1984) Nature 308, 693-698 (1985) J. Biol. Chem. 260,4661-4670 7. Rasmussen, H., and Barrett, P. Q. (1984) Physiol. Reu. 64,93829. Dixon, J. F., and Hokin, L. E. (1985) J. Bioi. Chem. 260,16068984 16071 8. Rittenhouse-Simmons, S. (1979) J. Clin. Invest. 6 3 , 580-587 30. Creutz, C. E., Dowling, L. G., Kyger, E. M., and Franson, R. C. 9. Kojima, I., Kojima, K., Kreutter, D., and Rasmussen, H. (1984) (1985) J. Biol. Chem. 2 6 0 , 7171-7173 J. Biol. Chem. 259, 14448-14457 31. Askoy, M. O., Murphy, R.A., and Kamm, K. E. (1982) Am. J. Physwl. 2 4 2 , C109-Cl16 10. Thomas, A. P., Marks, J. S., Coll, K. E., and Williamson, J. R. ~~

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