Mediated Hydrolysis of Phosphatidylcholine - Europe PMC

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Apr 4, 1991 - JIANGUO SONG,1 LAWRENCE M. PFIEFFER,2t AND DAVID A. FOSTERl*. Institute ...... Price, B. D., J. D. H. Morris, C. J. MarshaUl, and A. Hall.
MOLECULAR AND CELLULAR BIOLOGY, Oct. 1991, p. 4903-4908 0270-7306/91/104903-06$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 10

v-Src Increases Diacylglycerol Levels Via a Type D PhospholipaseMediated Hydrolysis of Phosphatidylcholine JIANGUO SONG,1 LAWRENCE M. PFIEFFER,2t AND DAVID A. FOSTERl* Institute for Biomolecular Structure and Function and Department of Biological Sciences, The Hunter College of The City University of New York, 695 Park Avenue,' and The Rockefeller University,2 New York, New York 10021 Received 4 April 1991/Accepted 4 July 1991

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with araehidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG pkroduction and. enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.

Protein kinase C (PKC) activity has been implicated in v-Src-induced intracellular signals (5, 12, 26, 33, 35, 37, 43). Diacylglycerol (DAG) is a second messenger that leads to the activation of PKC (25). DAG is generated from phospholipids either directly, through the action of type C phospholipases, or indirectly, through the action of type D phospholipases that generate phosphatidic acid (PA), which is then converted to DAG by a PA phosphatase (reviewed by Exton [10]). Increased levels of DAG have been observed in cells transformed by v-Src (22, 43); however, the source of the increased DAG is unclear. The time course for increased inositol phosphate levels in v-Src-transformed cells did not correlate with the time course for increased DAG (15, 22), suggesting that DAG produced in response to v-Src is generated from a source other than phosphatidylinositol (P1) or phosphorylated derivatives of PI. Over the last several years, considerable data have been presented implicating PI hydrolysis by type C phospholipases as the source of increased DAG levels in response to a variety of stimuli (reviewed by Berridge [2]). Although it is likely that type C phospholipase-mediated hydrolysis of PI constitutes a major signaling pathway leading to the production of biologically active inositol phosphates, it is not clear that increases in DAG levels seen in response to a variety stimuli can be accounted for by hydrolysis of this relatively minor phospholipid (10). In this regard, it has recently been demonstrated that phosphatidylcholine (PC) rather than PI is the major source of increased DAG production in response to a variety of stimuli (la, 3, 6, 14, 19, 20, 23, 30, 32, 42). In this report, we present evidence that increases in DAG observed in response to the protein-tyrosine kinase activity of v-Src are derived primarily from the hydrolysis of PC by a type D phospholipase and the subsequent hydrolysis of PA to DAG by a PA phosphatase.

MATERIALS AND METHODS Cells and cell culture conditions. BALB/c 3T3 and LA90-

transformed BALB/c 3T3 cells (provided by J. Brugge) were maintained in Dulbecco's modified Eagle medium supplemented with 10% newborn calf serum. Confluent cell cultures were made quiescent by maintaining cells for 2 days without a change in medium and then treating the cells with fresh medium containing 0.5% newborn calf serum overnight. Materials. [3H]myristate (NET-830), [3H]palmitate (NET043), [3H]arachidonate (NET-2982), [3H]choline (NET-109), [3H]inositol (NET-114A), and [3H]glycerol (NET-848H) were obtained from New England Nuclear. Propranalol, PI, PC, PA, DAG, and choline were obtained from Sigma. Thin-layer chromatography (TLC) plates (Silica Gel 60A) were from American Scientific Products. Phosphatidylethanol and phosphatidylbutanol were obtained from Avanti Polar Lipids. Prelabeling of phospholipids. Unless otherwise indicated, quiescent LA90 or BALB/c 3T3 cells in 60-mm culture dishes were prelabeled for 24 h in 2 ml of medium containing 0.5% newborn calf serum. Isotopes were added to the culture medium as follows: [3H]myristate, 10 ,uCi (40 Ci/ mmol); [3H]palmitate, 10 ,uCi (60 Ci/mmol); [3H]arachidonate, 2 ,uCi (240 Ci/mmol); [3H]choline, 5 ,uCi (85 Ci/mmol); [3H]inositol, 2 ,uCi (15 Ci/mmol); and [3H]glycerol, 20 ,uCi (40 Ci/mmol). Extraction of phospholipase products. (i) Lipids. Lipids were extracted as described by Billah et al. (4), with minor modifications. Prelabeled LA90 or BALB/c 3T3 cells were shifted from 40 to 35°C for the indicated times. Cells were then washed with isotonic Tris-saline buffer, rapidly treated with 0.6 ml of methanol (MeOH)-6 N HCl (50:2), and scraped from the culture dish. The MeOH-HCl-treated cells were then extracted with 0.6 ml of CHCl3. Phase separation was obtained by adding 200 ,ul of 1 M NaCl. The organic phase was recovered, dried under N2, and redissolved in CHCl3-MeOH (90:10).

* Corresponding author. t Present address: Department of Pathology, University of Tennessee College of Medicine, Memphis, TN 38163.

(ii) Choline and phosphocholine. Cells were washed and extracted as described above. H20 (200 ,ul) was added, and 4903

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the aqueous phase was recovered and dried by a Speed-Vac concentrator. The residue was redissolved in 50% MeOH. Characterization of phospholipid metabolites by TLC. Extracts of phospholipid metabolites were characterized by TLC as described by Billah et al. (4), with modifications. Lipid standards were visualized by treating TLC plates with iodine vapor. To quantitate metabolically labeled compounds, the appropriate regions of TLC plates were scraped and counted in a scintillation counter. The following solvent systems were used: for DAG, hexane-diethylether-MeOHglacial acetic acid (90:20:3:2); for PC and PI, CHCl3-MeOHglacial acetic acid-H20 (50:25:8:4); for phosphatidylethanol, phosphatidylbutanol, and PA, the upper phase of ethylacetate-trimethylpentane-acetic acid-H20 (90:50:20:100); and for choline and phosphocholine, 0.6% NaCl-MeOH-NH3 (50:50:1). DAG mass assay. The DAG mass assay was performed as described by Preiss et al. (31). Lipid extracts prepared as described above were labeled with bacterial DAG kinase using [y-32P]ATP, and the products were resolved by TLC using the solvent system described above for PA. Inositol phosphate analysis. Inositol phosphates were isolated as described by Paris and Pouysseguer (27) and separated by Amprep columns (Amersham) according to the vendor's instructions. RESULTS Activation of the kinase activity of v-Src leads to increased levels of DAG. Our previous studies demonstrated that v-Src activates PKC in both avian and murine fibroblasts (33, 37). Since PKC is activated by DAG (25), increases in DAG levels in response to the protein-tyrosine kinase of v-Src were examined. The effect of increased v-Src kinase activity was examined in BALB/c 3T3 cells infected with a temperature-sensitive strain of Rous sarcoma virus (LA90 cells, described by Gray and Macara [11] and Qureshi et al. [33]). Phospholipids of LA90 cells and the parental BALB/c 3T3 cells were metabolically prelabeled with [3H]glycerol for 24 h. Quiescent LA90 cells maintained at the nonpermissive temperature for v-Src (40°C) were shifted to the permissive temperature (35°C) to activate the protein-tyrosine kinase activity of v-Src, and levels of 3H-labeled DAG were examined by TLC. As shown in Fig. la, 3H-labeled DAG levels rapidly increased in LA90 cells after the temperature shift. This treatment had no effect on 3H-labeled DAG levels in the parental BALB/c 3T3 cell line (Fig. la). To establish that the increase in 3H-labeled DAG reflected real increases in DAG levels and not simply increases in [3H]glycerol incorporated into DAG in response to v-Src, we determined DAG levels in a DAG mass assay (31). As shown in Fig. lb, the temperature shift of LA90 cells led to an increase in DAG, as detected by phosphorylation of DAG to PA by bacterial DAG kinase. This effect was not seen in BALB/c 3T3 cells similarly treated. The time course for the generation of DAG was consistent with the time course for v-Src-induced PKC activity observed previdusly (33, 37). Differential labeling of phospholipids identifies the source of the v-Src-induced generation of DAG. Although increased levels of DAG in response to v-Src have been reported (22, 43), there have been conflicting reports on concomitant increases of inositol phosphates. Chiarugi et al. (8) reported a rapid increase in inositol phosphates in response to v-Src in rat fibroblasts; on the other hand, increased levels of inositol phosphates were not detected by Martins et al. (22) in chicken embryo fibroblasts or by Han et al. (13) in LA90-

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FIG. 1. v-Src-induced DAG. (a) LA90 and BALB/c 3T3 cells were prelabeled for 24 h with [3H]glycerol. Cells were shifted from the nonpermissive (40°C) to the permissive (34°C) temperature for v-Src, and levels of radioactively labeled DAG were determined at the times shown. Data are averages of three separate experiments. (b) DAG mass was determined by the method of Preiss et al. (31) in LA90 and BALB/c 3T3 cells after a temperature shift at the times shown. Data represent average increases in PA labeled with DAG kinase (as percentages of the control value) in two separate exper-

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transformed BALB/c 3T3 cells upon activation of v-Src. In our experiments, inositol phosphate levels did not change in response to v-Src in either avian or murine fibroblasts over the time period during which increased levels of DAG were observed (data not shown). Thus, it is unlikely that the increases in DAG shown in Fig. 1 are derived from PI or phosphorylated derivatives of PI. Phospholipids have characteristic fatty acid compositions (28, 29) that permit differential labeling with different radioactively labeled fatty acids (la, 7, 14, 21, 38, 39, 42). For example, PI is preferentially labeled with arachidonate, which is the predominant fatty acid esterified to the sn-2 position of the glycerol backbone of PI. In contrast, if cells are labeled with myristate, greater than 80% of the incorporated label is incorporated into PC. To determine the source of DAG produced in response to v-Src, we exploited this differential fatty acid composition of phospholipids. Table 1 shows the percentages of 3H-labeled myristate, arachidonate, palmitate, and glycerol incorporated into PC and PI in LA90 cells grown at 40°C. Myristate is incorporated almost exclusively into PC, with only about 2% of labeled myristate being incorporated into PI. Arachidonate is incorporated equally into both PC and PI; however, since PC is far more

VOL. 11, 1991

v-Src-INDUCED TYPE D PHOSPHOLIPASE a

TABLE 1. Differential labeling of phospholipids

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Label

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[3H]myristate [3H]arachidonate [3H]palmitate [3H]glycerol

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abundant (approximately 35% of the total phospholipid) than PI (approximately 3.5% of the total phospholipid) (9), the equivalent incorporation of arachidonate into PC and PI indicates that arachidonate is incorporated with high efficiency into PI. Glycerol and palmitate are incorporated into PC and PI with efficiencies reflecting the relative abundance of PI and PC. The differential efficiency of labeling phospholipids with fatty acid precursors (as shown in Table 1) was used to identify the source of v-Src-induced increases in DAG. When phospholipids were labeled with [3H]arachidonate, no increase in labeled DAG was detected in response to v-Src (Fig. 2). However, when either [3H]myristate or [3H]palmitate was used to prelabel phospholipids, increased DAG levels in response to v-Src were observed (Fig. 2). Since [3H]myristate and [3H]palmitate are efficiently incorporated into PC (Table 1) (14, 29), the data suggest that PC is the source of v-Src-induced DAG production. The lack of increased DAG in [3H]arachidonate-labeled cells suggests that the source of v-Src-induced DAG is not PI. v-Src induces production of choline with kinetics similar to

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FIG. 2. DAG production induced by v-Src detected after differential prelabeling with fatty acid precursors. LA90 cells were prelabeled with either [3H]myristate (3H-MYR), [3H]palmitate (3HPAL), or [3H]arachidonate (3H-AA) for 24 h. Cells were then shifted from the nonpermissive to the permissive temperature for v-Src, and DAG levels were determined as for Fig. 1. Data are averages of at least three independent experiments.

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FIG. 3. Evidence that activation of v-Src leads to increased choline production. Cells were prelabeled with [3H]choline for 24 h. Choline and phosphocholine were examined at the indicated times after a temperature shift from 40 to 35°C in LA90 cells (a) and BALB/c 3T3 cells (b). Data are averages of three independent experiments in LA90 cells and two independent experiments in BALB/c 3T3 cells.

those observed for DAG production. DAG can be generated by either type C or type D phospholipases. If v-Src-induced DAG is derived from PC, then phosphocholine would be produced if a type C phospholipase is activated, and choline would be produced if a type D phospholipase is activated. To observe changes in the level of choline metabolites, LA90 cells were metabolically labeled with [3H]choline and levels of intracellular choline and phosphocholine were measured by TLC after a shift from the nonpermissive to the permissive temperature. As shown in Fig. 3, increased levels of choline were detected in response to v-Src. Importantly, the time course of the increase was similar to that observed for DAG production. There was no change in phosphocholine levels in response to v-Src (Fig. 3). Although these data could also be explained by the activation of a phosphocholine phosphatase, to our knowledge no such activity has been reported in mammalian cells. These data provide additional evidence that PC is the source of the v-Srcinduced increase in DAG levels. Furthermore, since choline rather than phosphocholine was increased in response to v-Src, the data suggest that hydrolysis of PC results from the action of a type D phospholipase. Blocking of PA phosphatase inhibits v-Src-induced DAG. If, as suggested above, a type D phospholipase is activated by v-Src, then both choline and PA should be generated. However, consistent increases in the level of PA were not

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PRO + + FIG. 4. Effects of propranalol on v-Src-induced DAG, PA, and choline. LA90 cells were prelabeled with either [3H]palmitate or [3H]choline. Radioactivity (counts per minute) in DAG (a), PA (b), and choline (c) was examined before and 30 min after the shift to the permissive temperature for v-Src as described in Materials and Methods. The experiments were carried out in the presence and absence of propranalol (300 ,uM), which was added 5 min before the temperature shift. Cells were harvested 30 min after the temperature shift. Data are averages of duplicate cultures from a representative experiment using [3H]palmitate for DAG and PA measurements. Similar data were obtained with [3H]myristate used as a label.

detected in response to v-Src, possibly because of the rapid metabolic conversion of PA to DAG by a PA phosphatase. We therefore examined the effect of propranalol, which has been shown to inhibit PA phosphatase activity (4, 17), on v-Src-induced DAG, PA, and choline. Propranalol treatment of LA90 cells prior to activation of v-Src by temperature shift inhibited v-Src-induced increases in DAG (Fig. 4a). Concomitant with the loss of DAG production was the detection of an increase in PA levels in response to activat-

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FIG. 5. Stimulation of type D phospholipase by v-Src, as determined by transphosphatidylation activity. (a) LA90 cells were prelabeled with [3H]palmitate and shifted from the nonpermissive (40°C) to the permissive (35°C) temperature for v-Src in the presence and absence of either butanol (0.5%) or ethanol (0.1%). Radioactivity incorporated into phosphatidylbutanol (PBt) and phosphatidylethanol (PEt) separated by TLC was counted as described in Materials and Methods. (b) BALB/c 3T3 cells were prelabeled with [3H]palmitate and shifted from 40 to 35°C in the presence and absence of ethanol (0.1%), and levels of radioactivity incorporated into phosphatidylethanol were determined as for panel a.

ing v-Src (Fig. 4b). Propranalol treatment had little effect on choline production induced by v-Src (Fig. 4c). Although the brief treatment with propranalol may induce effects other than inhibition of PA phosphatase activity, the data demonstrate that v-Src is able to induce the production of both PA and choline, the expected products of a type D phospholipase-mediated hydrolysis of PC. v-Src induces transphosphatidylation activity. Type D phospholipases catalyze the transphosphatidylation of PC to phosphatidylethanol or phosphatidylbutanol in the presence of ethanol or butanol (14, 16, 34). This assay has been used to distinguish between type C and type D phospholipases (14). Activation of v-Src by temperature shift of LA90 cells in the presence of 0.1% ethanol or 0.5% butanol led to substantial increases in phosphatidylethanol and phosphatidylbutanol (Fig. Sa). No change in phosphatidylethanol was seen in BALB/c 3T3 cells upon the temperature shift (Fig. Sb). These data demonstrate that v-Src induces type D phospholipase activity and further suggest that v-Src-induced increases in DAG result from type D phospholipasemediated hydrolysis of phosphatidylcholine.

v-Src-INDUCED TYPE D PHOS,-IOLIPASE

VOL . 1 l, 1991

DISCUSSION We have shown that v-Src-induced increases in DAG derive from a cellular lipid that is preferentially labeled with myristate. Myristate is preferentially incorporated into PC, marking PC as the source of v-Src-induced DAG. Consistent with this conclusion, we found that free choline levels were elevated in response to the kinase activity of v-Src. The generation of choline rather than phosphocholine suggests a type D phospholipase/PA phosphatase mechanism for generating DAG. Consistent with this hypothesis, we found that blocking PA phosphatase with propranalol inhibited v-Srcinduced increases in DAG levels and led to concomitant v-Src-induced increases in PA. Finally, activating the protein-tyrosine kinase activity of v-Src increased transphosphatidylation activity which is catalyzed by type D phospholipases. Taken together, the data presented here are all consistent with the hypothesis that v-Src-induced increases in DAG derive from PC via a type D phospholipase and PA phosphatase. Increased levels of inositol phosphates in v-Src-transformed rat cells have been reported (8, 15). Although increased levels of inositol phosphates in response to v-Src were not detected in LA90 cells concomitant with the production of DAG, it is still possible that low levels of DAG could be derived from PI. However, if some DAG is generated from PI, the contribution to total DAG produced in response to v-Src is small relative to that generated from PC, since the DAG we observe in response to v-Src is derived primarily from a phospholipid into which myristate is efficiently incorporated. Myristate is not incorporated into PI (Table 1). In addition, v-Src-induced DAG was not detected when cells were labeled with a fatty acid (arachidonate) which is preferentially incorporated into PI (Fig. 2). The failure of LA90 v-Src to induce detectable PI turnover in BALB/c 3T3 cells while inducing an increase in DAG is similar to the effect obtained with temperature-sensitive NY74-2 v-Src in infected chick cells (22). DAG generated from PC could be responsible for the activation of PKC by v-Src. However, PKC-dependent activation of type D phospholipase-mediated hydrolysis of PC has been reported (7, 14, 23, 32). The data presented here do not establish whether PKC activation is required for v-Src-induced PC hydrolysis or a consequence of v-Srcinduced PC hydrolysis; however, the kinetics of v-Srcinduced DAG production correlates with v-Src-induced phosphorylation of the PKC substrate MARCKS (33, 37); therefore, the DAG produced from PC hydrolysis likely contributes to the activation of PKC by v-Src. The transphosphatidylation activity induced by v-Src suggests that a PC-specific type D phospholipase is the signaling component that is activated by v-Src; however, PA phosphatase could also be activated by v-Src. The mechanism by which v-Src induces a type D phospholipase/PA phosphatase mechanism for increasing DAG levels is not yet clear. Preliminary data from our laboratory suggest that a G protein is required for v-Src to activate PKC (1). Thus, v-Src-induced PC hydrolysis may be indirect and require the activation of a G protein which activates a PC-specific type D phospholipase to generate the DAG that in turn activates PKC. To generate DAG via a type D phospholipase requires a second enzyme (a PA phosphatase) to convert PA to DAG. This raises a question as to what advantage there is in using an indirect pathway to increase DAG levels when DAG levels can be increased directly by using type C phospholi-

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pases. Other protein-tyrosine kinases have been demonstrated to be associated with phospholipase C--y, which hydrolyzes PI-4,5-bisphosphate (18, 24). The induction of a type D phospholipase instead of a type C phospholipase by v-Src to increase DAG levels may generate a more complex or specific biological signal. In this regard, the generation of phosphatidic acid may be important. It has been suggested that PA or more likely a metabolite of PA inhibits the H-Ras GTPase inhibitory protein, GAP (40, 44). Thus, the production of PA might enhance v-Src signals going through H-Ras, which has previously been implicated in v-Src signaling (36). Another possibility for generating PA would be to provide the metabolic precursor for the mitogenic phospholipid lysophosphatidic acid, which is the product of type A2 phospholipase-mediated hydrolysis of PA (41). Consistent with the possibility that lysophosphatidic acid may be generated from v-Src-induced PA, increases in the level of monoacylglycerol (the primary metabolite of lysophosphatidic acid) in response to v-Src have been detected (36a). The difficulty in detecting PA production in the absence of propranalol is consistent with the suggestion that PA is converted into biologically active molecules other than DAG, such as lysophosphatidic acid. Thus, an indirect method for increasing DAG levels may be important for the induction of more complex biological signals which could include the positive stimulation of H-Ras-mediated signals and the production of the mitogenic phospholipid lysophosphatidic acid. ACKNOWLEDGMENTS We thank Joan Brugge for the LA90 cells. Sajiad Qureshi is acknowledged for many helpful discussions and for establishing the LA90 system in our laboratory. We thank Tom Haines and Rudolph Spangler for valuable comments on the manuscript. This investigation was supported by National Institutes of Health grant CA46677 and PSC-CUNY research award 669115 to D.A.F. and a Research Centers in Minority Institutions award from the Division of Research Resources, National Institutes of Health (RRO-3037-03), to Hunter College. REFERENCES 1. Alexanandropoulos, K., C. K. Joseph, R. Spangler, and D. A. Foster. 1991. Evidence that a G-protein transduces signals initiated by the protein-tyrosine kinase v-Fps. J. Biol. Chem.

266:15583-15586. la.Augert, G., S. B. Bocckino, P. F. Blackmnore, and J. H. Exton. 1990. Hormonal stimulation of diacylglycerol formation in hepatocytes. J. Biol. Chem. 264:21689-21698. 2. Berridge, M. J. 1987. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56: 159-194. 3. Besterman, J. M., V. Duronio, and P. Cuatrecasas. 1986. Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger. Proc. Natl. Acad. Sci. USA 83:6785-6789. 4. Billah, M. M., S. Eckel, T. J. Mullmann, R. W. Egan, and M. I. Siegel. 1990. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. J. Biol. Chem. 264: 17069-17077. 5. Blobel, G., and H. Hanafusa. 1991. The v-src inducible gene 9E3/pCEF4 is regulated by both its promoter upstream sequence and its 3' untranslated region. Proc. Natl. Acad. Sci. USA 88:1162-1166. 6. Bocckino, S. B., P. F. Blackmore, and J. H. Exton. 1985. Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine and angiotensin II. J. Biol. Chem. 260:14201-14207. 7. Cabot, M. C., C. J. Welsh, H.-T. Cao, and H. Chabbott. 1988.

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