0013-7227/90/1273-1436$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
A Potential Role for Phospholipase-D in the Angiotensin II-Induced Stimulation of Aldosterone Secretion from Bovine Adrenal Glomerulosa Cells* WENDY B. BOLLAGt, PAULA Q. BARRETT, CARLOS M. ISALES, MORDECHAI LISCOVITCH$, AND HOWARD RASMUSSEN Departments of Cellular and Molecular Physiology and Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510; and the Department of Hormone Research, Weizmann Institute of Science, Rehovot 76100, Israel
only a modest effect on aldosterone production, the stimulatory action of PLD was enhanced, yielding a secretory rate (442 ± 119 pg/min-mg protein) that was approximately 60% of that elicited by 10 nM Ang II (763 ± 182 pg/min-mg protein). Exogenous PLD also induced a significant increase in DAG levels (from 0.76 ± 0.03 to 1.10 ± 0.1 nmol/mg protein), which was not altered by the addition of Bay K 8644. However, PLD did not stimulate inositol phosphate production. These data indicate that 1) Ang II activates PLD; 2) exogenous PLD can elevate aldosterone secretory rates and DAG levels without eliciting phosphoinositide hydrolysis; and 3) the stimulatory action of exogenous PLD on aldosterone secretion is enhanced in the presence of Bay K 8644. Thus, PLD-induced DAG production may play an important role in the Ang II-mediated stimulation of aldosterone secretion from the adrenal zona glomerulosa. (Endocrinology 127: 1436-1443, 1990)
ABSTRACT. The mechanism by which angiotensin-II (Ang II) stimulates aldosterone secretion from adrenal glomerulosa cells involves a phospholipase-C-mediated increase in phosphoinositide turnover and diacylglycerol (DAG) production. Because agonist-induced activation of phospholipase-D (PLD) also contributes to elevations in DAG in other cell types, the ability of Ang II to stimulate PLD activity in cultured bovine adrenal glomerulosa cells was examined. Ang II elicited significant increases in the levels of phosphatidic acid and, in the presence of ethanol, of phosphatidylethanol, a more specific marker for PLD activation. The potential role of this increased PLD activity in the regulation of aldosterone secretion was examined by investigating the ability of exogenous PLD to alter secretory rates. PLD alone dose-dependently increased aldosterone secretion from 5.9 ± 0.5 to 135 ± 48 pg/min-mg protein. In the presence of the calcium channel agonist Bay K 8644, which by itself had
U
PON binding to their receptors many hormones activate phospholipase-C (PLC) to produce, via the hydrolysis of phosphatidylinositol 4,5-bisphosphate, two second messengers: inositol 1,4,5-trisphosphate (Ins1,4,5-P3) and diacylglycerol (DAG) (1,2). Data from this laboratory have been interpreted in terms of a model of hormone action in which the Ins-l,4,5-P3-induced release of calcium from an intracellular storage site initiates the cellular response, and an elevation in membrane DAG content, in conjunction with a hormone-elicited increase in calcium influx, underlies sustained hormonal responses (3-5). Recent evidence, however, suggests that Received April 9,1990. Address requests for reprints to: Dr. Howard Rasmussen, Department of Internal Medicine, Yale University School of Medicine, P.O. Box 3333, New Haven, Connecticutt 06510. * This work was supported in part by NIH Grants DK-19813 (to H.R.), HL-36977 (to P.Q.B.), and GM-07527 (to W.B.B.). This work was performed in partial fulfillment of the requirements for a Doctorate of Philosophy (W.B.B.). t Recipient of a predoctoral fellowship from the NSF. X Recipient of a travel grant from the Yale University-Weizmann Institute Collaboration Program.
an increase in PLC activity may not be the sole pathway by which hormones increase membrane DAG content (6, 7). In a number of systems phospholipase-D (PLD) has also been implicated in the agonist-induced elevation of DAG (8-15). The phosphatidic acid (PA) produced by this enzyme may be converted to DAG via the action of phosphatidate hydrolases (7). In hepatocytes, vasopressin elicits alterations in both the levels and the fatty acid composition of PA, and these changes in PA precede similar changes in DAG (8). These results indicate that the activation of PLD, rather than the phosphorylation of PLC-generated DAG, is likely to be responsible for the observed PA production. Similarly, stimulation of astrocytoma cells with carbachol results in a pattern of radiolabeled choline and phosphorycholine production which suggests that phosphatidylcholine (PC) is hydrolyzed by a PLD- as well as a PC-specific PLC (12). Moreover, in neutrophils prelabeled with 3Hor 32P-labeled l-O-hexadecyl-2-acyl-src-glycero-3-phosphocholine and stimulated with /-methionyl-leucylphenyl-alanine (fMLP), a comparison of the ratio of 3H
1436
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:19 For personal use only. No other uses without permission. . All rights reserved.
PLD AND ALDOSTERONE SECRETION to 32 P in l-O-alkyl-2-acyl-PA suggests that PLD is involved in the measured increase in l-O-alkyl-2-acyl-PA production (9). In addition, treatment of HL-60 granulocytes with fMLP results in the generation of PA and, in the presence of ethanol, phosphatidylethanol (PEt), a marker for PLD activation (10, 13, 14). The production of P E t has also been demonstrated in several cell types in response to hormonal stimulation (11, 15, 16). Thus, accumulating evidence points to a potential role for PLD in the response of cells to physiological stimuli as well as in the increase in membrane DAG content elicited by various hormones. In adrenal glomerulosa (AG) cells, angiotensin-II (Ang II) stimulates phosphoinositide (PI) turnover with the resultant production of Ins-1,4,5-P 3 and DAG (3, 17-21). Studies in bovine glomerulosa cells have provided evidence that DAG is important in the sustained aldosterone secretory response induced by Ang II (3, 22-24). In light of the reported contributions of PLD activation to the elevation in DAG levels in other systems, we investigated the effect of Ang II on PLD activity and the potential ability of PLD-stimulated PA production to elevate DAG levels and affect the aldosterone secretory rate. We found that in cultured bovine AG cells Ang II stimulated PLD activity, and an exogenously added bacterial PLD elicited a secretory response that was synergistic with the calcium channel agonist Bay K 8644. These actions were related to an effect of the enzyme on membrane DAG content and not to an effect of PLD on total intracellular inositol phosphates. Thus, our results indicate that stimulation of PLD activity can result in an elevation in membrane DAG content and suggest the possible involvement of endogenous PLD in the aldosterone secretory response of AG cells to hormones such as Ang II. Materials and Methods Bovine AG cell isolation and culture Bovine AG cells were isolated as described previously (25). Briefly, glomerulosa cell slices were prepared from adrenal glands obtained from a local slaughterhouse, and AG cells were dispersed from collagenase-digested slices by mechanical agitation. Freshly isolated AG cells were purified on a 56% Percoll gradient and cultured overnight in Falcon Primaria dishes (Becton Dickinson Labware, Lincoln Park, NJ) in a Dulbecco's Modified Eagle's Medium-Ham's F-12 medium (1:1) containing 10% horse serum (vol/vol), 2% fetal bovine serum (vol/vol), ascorbate (100 fiM), cv-tocopherol (1.2 pM), Na 2 Se0 3 (0.05 juM), butylated hydroxyanisole (50 JUM), metyrapone (5 HM), penicillin (100 U/ml), streptomycin (100 fig/ml), gentamycin (30 ng/ ml), and amphotericin-B (3 Mg/ml), as previously described (26). Serum-containing medium was then removed and replaced with serum-free medium (with 0.2% BSA with or without [3H] inositol or [3H]oleic acid), and the cells were incubated for an
1437
additional 20-24 h before use. In some experiments ticarcillin and amikacin (200 ixg/m\) were substituted for gentamycin. Secretion experiments Before stimulation, cells were incubated at 37 C in KrebsRinger bicarbonate (KRB) buffer (120 mM NaCl, 24.9 mM NaHCO3, 3.5 mM KC1, 1.2 mM MgSO4, 1.2 mM NaH2PO4) 1.25 mM CaCl2, 0.1% dextrose, and 0.2% BSA) gassed with 5% CO2 (and containing also 2.5 mM Na acetate and 0.002% phenol red) for a period of 2 h. The supernatants were collected for measurement of control aldosterone secretory rates, and KRB containing the appropriate agents was added. After a 2-h stimulation period, supernatants were collected. In time-course experiments, after a 30-min incubation, the supernatants were collected for determination of control secretory rates, and the various agonists were added. Thereafter, at the indicated times (every 10 or 20 min) supernatants were collected and replaced with KRB containing the appropriate agonist. All supernatants were stored at —20 C until assayed. At the conclusion of each experiment, cells were solubilized in 0.4 N NaOH and stored at —20 C for protein determination. Determination of membrane DAG content Cells were preincubated at 37 C in KRB (plus 2.5 mM Na acetate and 0.002% phenol red) for 30 min in 5% CO2. The appropriate agents were added, and the incubations were continued for an additional 30 min. The stimulation period was terminated by the addition of 0.2% sodium dodecyl sulfate (SDS), and neutral lipids were extracted essentially according to the method of Bligh and Dyer (27). Briefly, the SDSsolubilized cells were extracted with ice-cold chloroform-methanol (1:2, vol/vol) for 1-2 h. Additional chloroform was added, and the lower phases were collected and washed with methanol0.2 M NaCl. The washed lower phases were dried under N2, stored at -20 C, and assayed within 72 h, using a slight modification of the DAG kinase method of Preiss et al. (28). As described previously (20), an Escherichia coli DAG kinase preparation obtained from Lipidex (Middletown, WI) was employed to phosphorylate DAG to PA using adenosine 5'-[Y- 3 2 P] triphosphate. PA was extracted into chloroform and chromatographed on a silica gel TLC plate (Merck, Darmstadt, West Germany) using a solvent system of chloroform-acetone-methanol-acetic acid-water (50:20:10:10:5, vol/vol/vol/vol/vol). After autoradiography, the spot corresponding to PA was placed in scintillation fluid, and radioactivity was determined in a liquid scintillation spectrometer (Packard, Sterling, VA). Measurements of total radiolabeled inositol phosphates After a 20- to 24-h labeling period in serum-free medium containing 0.2% BSA and 5 /ttCi/ml [3H]inositol as described above, cells were washed and preincubated for 30 min at 37 C in KRB. After the addition of the appropriate agents, the incubation was continued for 30 min and terminated with 10% perchloric acid (PCA). The PCA extract was collected and perchlorate precipitated with KOH. The supernatants were loaded onto Dowex AG1-X8 (Bio-Rad, Richmond, CA) columns, which were then washed with water and 5 mM Borax-60
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:19 For personal use only. No other uses without permission. . All rights reserved.
PLD AND ALDOSTERONE SECRETION
1438
mM Na formate to remove radiolabeled inositol and glycerophosphoinositol, respectively. Total inositol phosphates were subsequently eluted using 1.0 M ammonium formate-0.1 M formic acid. Aliquots were mixed with scintillation fluid and quantified. The PCA-precipitated cells were solubilized in 0.4 N NaOH and stored at -20 C for protein determination. Measurements of PA and PEt production Cells were labeled in serum-free medium contaning 0.2% BSA and 5 MCI/HII [3H]oleic acid for 20-24 h. After washing and a 30-min preincubation period, cells were stimulated with the appropriate agonists and incubated for an additional 30 min in the presence of 0.5% ethanol. The cells were then solubilized in 0.2% SDS. Small aliquots of the SDS-solubilized cells were saved for protein determination, and the remainder was extracted for 1-2 h with ice-cold chloroform-methanol to which acetic acid and 10 mM EGTA, pH 7.2 (1:2:0.04:0.2, vol/ vol/vol/vol) were added. Additional chloroform and 0.2 M NaCl were added to break phase, and the lower phases were collected and dried under N2. Samples were resuspended in chloroformmethanol and spotted onto heat-activated silica gel 60 TLC plates (0.25 mm thickness with concentrating zone). Plates were developed in a solvent system of ethyl acetate-isooctaneacetic acid-water (13:2:3:10) and were visualized with autoradiography using En3Hance (New England Nuclear, Boston, MA). Spots corresponding to PA and PEt, identified by comigration with authentic standards, were placed in liquid scintillation fluid and quantified. [3H]Oleate was chosen to monitor the formation of PA and PEt because this fatty acid is a major constituent and, thus, labels the phosphatidyl moiety of all phospholipids. Therefore, using this radiolabel PLD activity can be monitored regardless of the identity of the phospholipid that serves as the substrate of the enzyme. Materials Collagenase was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Myo-[2-3H]inositol, [9,10-n-3H] oleic acid, and adenosine 5'-[7-32P]triphosphate were obtained from Amersham (Arlington Heights, IL). DAG kinase was purchased from Lipidex (Middletown, WI), and Percoll from Pharmacia (Piscataway, NJ). Fetal bovine serum, horse serum, penicillin, and streptomycin were obtained from Gibco (Grand Island, NY); all other tissue culture reagents were from Sigma (St. Louis, MO). PLD from Streptomyces chromofuscus was also purchased from Sigma. One international unit will hydrolyze 1.0 jumol choline from L-a-PC (egg yolk) per min at pH 8.0 and 30 C. Bay K 8644 was a generous gift from Dr. Alexander Scriabine, Miles Institute for Preclinical Pharmacology (West Haven, CT). Aldosterone was assayed using a solid phase RIA (Diagnostic Products, Los Angeles, CA). PEt standard was prepared according to the method of Eibl and Kovatchev (29). Protein was determined by the method of Lowry et al. (30), using BSA as standard.
Results Activation of PLD by Ang II As reported in other systems, the activation of PLD would be expected to increase PA production and, in the
Endo• 1990 Vol 127 • No 3
presence of ethanol, generate PEt, a unique phospholipid that is characteristically produced by PLD activity when ethanol acts as a phosphatidyl group acceptor (31). In bovine AG cells prelabeled with [3H]oleic acid for 20-24 h, a 30-min incubation with Ang II (10 nM) resulted in an approximately 75% increase in radiolabeled PA, as shown in Fig. 1. More importantly, in the presence of ethanol, PEt production was also increased 82% by Ang II stimulation. These data suggest that Ang II activates PLD, resulting in the production of PA. This PLDcatalyzed formation of PA could, via the activity of PA hydrolases, provide a source of DAG in addition to that generated by Ang II-induced activation of PLC. Effect of exogenous PLD on aldosterone secretion If PLD is activated by Ang II in situ, then exogenous PLD might also be expected to stimulate aldosterone secretion from AG cells by providing one of the signals mediating hormonal action. In fact, exogenous PLD increased the levels of [3H]oleate-labeled PA from a control value of 3,799 ± 224 to 16,096 ± 2,047 cpm/mg protein (P < 0.0005), as well as the levels of radiolabeled PEt (control, 2,445 ± 145; PLD, 3,318 ± 371 cpm/mg protein; P < 0.05). As illustrated in Fig. 2, exogenous PLD also elicited small dose-dependent increases in aldosterone secretion. The PLD-stimulated secretory response attained a maximal value at a PLD concentration of 0.1 IU/ml (a concentration at which 0.1 ;umol PA/min are liberated from PC at pH 8.0 and 30 C). A further increase in the PLD concentration, however, resulted in a decrease in aldosterone secretion to 51 pg/min-mg protein from the peak rate of 135 pg/min • mg protein, generating in essence a bell-shaped curve. However, enzyme that had been inactivated by boiling did not stimulate aldosterone secretion (data not shown). The secretory rates depicted in Fig. 2A are the mean values obtained in three separate experiments. The relatively large SEs, which tend to obscure the dose dependence of the PLD-induced aldosterone secretory response, are the result of large differences between, rather than within, experiments. Thus, in a single experiment with a control secretory rate of 10.0 ± 0.4 pg/min • mg protein, a dose of 0.005 IU/ml PLD elicited a secretory rate of 210.3 ± 0.5 pg/min-mg protein, 0.01 IU/ml elicited a rate of 212.4 ± 4.0, 0.05 IU/ml elicited a rate of 243.5 ± 13.0, 0.1 IU/ml elicited a rate of 282.5 ± 4.2, and 1.0 IU/ ml elicited a rate of 99.3 ± 24.1 pg/min-mg protein. In comparison to the Ang II-induced increase in aldosterone secretion (to 1155.9 ± 186.8 pg/min-mg protein), however, these dose-dependent changes in steroidogenesis are quite modest. Nevertheless, exogenous PLD should generate only one (DAG) of the signals thought to be involved in the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:19 For personal use only. No other uses without permission. . All rights reserved.
PLD AND ALDOSTERONE SECRETION
1439
8000
FIG. 1. Ang II increases the levels of radiolabeled PA and PEt. Cultured bovine AG cells were prelabeled for 20-24 h with 10 fid [3H]oleic acid and stimulated for 30 min with 10 nM Ang II in the presence of 0.5% ethanol. Phospholipids were extracted and chromatographed as described in Materials and MethodsM, Radioactivity found in PA; §, radioactivity found in PEt. Values represent the means (±SE) of five determinations from two separate experiments. *, P < 0.002 us. control (CON).
TO Q.
6000 -
5 2
4000 -
2000 -
CON
ANG II
1000 -
800 -
o>
(0
k.
o
a> o i_
o
600 -
Q.
Co
1.0-
CO . i .
5 0.5-
0.0 CON
ANG II
PLD
PLD+BAY K BAY K
FIG. 4. Exogenous PLD elevates DAG levels. AG cells were stimulated for 30 min with 10 nM ANG II, 0.1 IU/ml PLD, 100 nM Bay K 8644, or PLD plus Bay K 8644. DAG levels were measured by a DAG kinase method and are expressed as fold over control values. Control values were 0.76 ± 0.03 nmol DAG/mg protein. Data points are the mean (± SE) of four determinations from two separate experiments. *, P < 0.025 us. control (CON). TABLE 1. Total [3H]inositol phosphates produced in response to exogenous PLD, Bay K 8644, and Ang II Inositol phosphates (cpm/mg protein)0
Agonist Control Bay K 8644 (100 nM) PLD (0.1 IU/ml) PLD + Bay K 8644 Ang II (10 nM)
3,568 ± 370 3,256 ± 292 4,161 ± 348 5,040 ± 1,276 26,573 ± 1.8176 0 Values represent the mean ± SE of a minimum of four determinations from at least two separate experiments. 6 P < 0.0001 vs. control.
mg protein; P < 0.0001).
Discussion In view of recent reports demonstrating the activation of PLD in response to a variety of hormones (8-15), we examined the ability of Ang II to stimulate PLD activity in cultured bovine AG cells. In the presence of ethanol, PLD catalyzes not only the production of PA, but also, as a result of its intrinsic phosphatidyl transferase activity, the generation of PEt. Numerous investigators have demonstrated the validity of assessing PLD activation using PEt production as an assay (10, 11, 13-16). Our finding that Ang II increased levels of radiolabeled PA and PEt thus constitutes strong evidence that this hormone activates PLD. The result of such activation would be the production of PA from which DAG could be
1441
generated by the action of phosphatidate hydrolases, if such enzymes were present and active in AG cells. We have previously proposed (2, 22, 32) that the Ang II-elicited increase in membrane DAG content in combination with an enhanced calcium influx underlies sustained aldosterone secretion in bovine AG cells (2, 22, 32). Thus, if phosphatidate hydrolases were functioning in these cells, their activity toward PLD-generated PA could result in DAG production and a potential increase in the aldosterone secretory response. We, therefore, investigated the effect of exogenous PLD on the secretion of aldosterone from cultured bovine AG cells. We found that exogenous PLD stimulated secretion, and that this effect was enhanced by Bay K 8644 (Figs. 2 and 3). This result is similar to the synergism observed between Bay K 8644 and phorbol esters (22), the latter of which are thought to substitute for the physiological signal DAG. Together these results suggest that the action of PLD to generate PA ultimately produced DAG via the activity of phosphatidate hydrolases on PLD-generated PA. Our results, however, raise the question of how the PA generated by the enzyme at the outer leaflet of the plasma membrane obtained access to the inner leaflet and to intracellular enzymes in order to increase aldosterone biosynthesis. It is well known that phospholipid "flipflop" within the lipid bilayer is extremely slow, and presumably PA, with its polar phosphate group, might have difficulty traversing the membrane. Pagano and Longmuir (33) have characterized the movement of a fluorescent PA analog within the lipid bilayer and have demonstrated that this probe is able to gain access to intracellular compartments. They further showed that this intracellular movement is the result of conversion of the PA to DAG. DAG, lacking a polar headgroup, would be expected to move freely within the plasma membrane. Indeed, in erythrocyte membranes, DAG has been reported to flip-flop within 1 min of its generation by an exogenous PLC (34), while in vesicles, transbilayer movement of a DAG analog was complete within 15 sec (35). We have demonstrated that the production of PA by exogenous PLD also results in an elevation in DAG levels, which functionally may be coupled to an increase in aldosterone secretion. Therefore, it appears likely that the action of PLD on the phospholipids of the outer leaflet results in the production of outer leaflet PA, which, after conversion to DAG, can easily traverse the membrane to activate intracellular enzymes. However, excessive phospholipid hydrolysis and DAG production with higher concentrations of PLD may alter membrane curvature (34), fluidity, or potential or in other ways damage the cells to yield the observed bell-shaped dose response to exogenous PLD. One intracellular enzyme whose activity may be enhanced by DAG is protein kinase-C (PKC), the phos-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:19 For personal use only. No other uses without permission. . All rights reserved.
1442
PLD AND ALDOSTERONE SECRETION
pholipid-dependent calcium-sensitive DAG-activated protein kinase (36). In bovine AG cells, PKC has been implicated in Ang II-elicited steroidogenesis, as PKC activators, such as phorbol esters and synthetic DAGs, mimic the effect of Ang II on both steroidogenesis and protein phosphorylation when used in conjunction with agents that enhance calcium influx (22, 24). The Ang IIinduced translocation of PKC from the cytosol to the plasma membrane also suggests a potential role for PKC in the regulation of aldosterone biosynthesis in the bovine cell (37). In this report we demonstrated that PLD stimulated aldosterone secretion by a mechanism presumably related to its ability to elevate DAG levels, and this stimulation of aldosterone secretion was enhanced by Bay K 8644. In fact, the combination of PLD and Bay K 8644 produced an aldosterone secretory response similar to that elicited by the calcium channel agonist used in conjunction with a synthetic DAG, oleoylacylglycerol (22). In both cases (22 and Table 1) there is no evidence that the agonists induced an increase in the hydrolysis of phosphoinositides. Together these results provide further evidence for PKC involvement in the control of Ang II-elicited increases in aldosterone secretion. Although we propose here that the PA generated by PLD activity serves as a precursor for DAG, other investigators have suggested a direct second messenger role for PA (6, 7, 11). Our results do not exclude such a possibility. Nevertheless, the observed aldosterone secretory response to exogenous PLD and Bay K 8644 is very similar to the synergism reported between agents that enhance calcium influx and phorbol esters or synthetic DAGs (22). Since the latter are presumed to function as substitutes for DAG in the activation of PKC, we interpret the similarities between the responses to these agents and exogenous PLD to indicate that PLD increases aldosterone secretion by providing PA, a DAG precursor. Although the data presented here were obtained in cultured cells, these cells are similar in most respects to the freshly isolated cells we have previously characterized. They are stimulated to secrete aldosterone by a variety of agonists, including Ang II, ACTH and carbachol (unpublished observations), and are inhibited by atrial natriuretic peptide (26). In addition, in these cultured cells Ang II elicits both a transient increase in cytosolic calcium (38) and an elevation in DAG content. Moreover, the aldosterone secretory responses to increasing concentrations of Ang II are similar in the cultured and freshly isolated cells (unpublished observations). Thus, the results obtained in this study are likely to be applicable to freshly dispersed AG cells as well. Our results provide evidence that active phosphatidate
Endo • 1990 Vol 127* No 3
hydrolases were present in cultured AG cells and that their activity toward PLD-generated PA could elevate DAG levels. In turn, this elevation resulted in an increase in the aldosterone secretory rate, presumably via an activation of PKC. The demonstration that Ang II activated an endogenous PLD, therefore, suggested a potential mechanism for the Ang II-elicited production of DAG in addition to PLC-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate. Because PLD activity may be calcium independent (39), a PLD/phosphatidate hydrolase mechanism of DAG generation with prolonged Ang II exposure may provide an explanation for the recent observation that sustained DAG production in AG cells is relatively insensitive to decreased extracellular calcium concentrations (21). In addition, PLD has been reported to hydrolyze preferentially PC, exhibiting more than a 30-fold greater activity toward this phospholipid than towards phosphatidylinositol, sphingomyelin, or phosphatidylethanolamine (39). Thus, different substrate specificities of PLC us. PLD may also provide a mechanism for the recently suggested multiplicity of sources of DAG in Ang II-stimulated AG cells (21). In turn, the hydrolysis of different phospholipids would presumably generate multiple DAG species, which could potentially play specific roles in the regulation of PKC activity and aldosterone secretion. Together these data indicate that not only are phosphatidate hydrolases present in the AG cell, but their activity on PLD-generated PA can result in the production of DAG. In addition, these hydrolases may be physiologically relevant in the response of the glomerulosa cell to Ang II, since this hormone appears to activate endogenous PLD to generate PA. Thus, Ang II-elicited stimulation of this endogenous PLD could provide a source of DAG in addition to the PLC-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate.
Acknowledgments We would like to express our appreciation for the expert technical assistance of Laura Kiernan and John Nee.
References 1. Berridge MJ 1987 Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56:159 2. Rasmussen H, Barrett P, Apfeldorf W, Takuwa Y, Takuwa N, Smallwood J, Isales C, Bollag W, Stein P 1987 Multiplicity of Ca2+ signal transduction pathways. In: Heilmann C (ed) Calcium-Dependent Processes in the Liver. MTP Press, Norwell, p 9 3. Kojima I, Kojima K, Kreutter D, Rasmussen H 1984 The temporal integration of the aldosterone secretory response occurs via two intracellular pathways. J Biol Chem 259:14448 4. Rasmussen H 1986 The calcium messenger system. N Engl J Med 314:1094 5. Rasmussen H 1986 The calcium messenger system. N Engl J Med 314:1164 6. Loffelholz K 1989 Receptor regulation of choline phospholipid hydrolysis. Biochem Pharmacol 38:1543
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:19 For personal use only. No other uses without permission. . All rights reserved.
PLD AND ALDOSTERONE SECRETION 7. Exton JH 1990 Signaling through phosphatidylcholine breakdown. J Biol Chem 265:1 8. Bocckino SB, Blackmore PF, Wilson PB, Exton JH 1987 Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J Biol Chem 262:15309 9. Agwu DE, McPhail LC, Chabot MC, Daniel LW, Wykle RL, McCall CE 1989 Choline-linked phosphoglycerides. J Biol Chem 264:1405 10. Billah MM, Pai J-K, Mullman TJ, Egan RW, Siegel MI 1989 Regulation of phospholipase D in HL-60 granulocytes. J Biol Chem 264:9069 11. Liscovitch M, Amsterdam A 1989 Gonadotropin-releasing hormone activates phosphlipase D in ovarian granulosa cells. J Biol Chem 264:11762 12. Martinson EA, Goldstein D, Brown JH 1989 Muscarinic receptor activation of phosphatidylcholine hydrolysis. J Biol Chem 264:14748 13. Pai J-K, Siegel MI, Egan RW, Billah MM 1988 Activation of phospholipase D by chemotactic peptide in HL-60 granulocytes. Biochem Biophys Res Commun 150:355 14. Pai J-K, Siegel MI, Egan RW, Billah MM 1988 Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. J Biol Chem 263:12472 15. Rubin R 1988 Phosphatidylethanol formation in human platelets: evidence for thrombin-induced activation of phospholipase D. Biochem Biophys Res Commun 156:1090 16. Liscovitch M 1989 Phosphatidylethanol biosynthesis in ethanolexposed NG108-15 neuroblastoma x glioma hybrid cells. J Biol Chem 264:1450 17. Balla T, Baukal AJ, Guillemette G, Morgan RO, Catt KJ 1986 Angiotensin-stimulated production of inositol trisphosphate isomers and metabolism through inositol 4-monophosphate in adrenal glomerulosa cells. Proc Natl Acad Sci USA 83:9323 18. Balla T, Baukal AJ, Guillemette G, Catt KJ 1988 Multiple pathways of inositol polyphosphate metabolism in angiotensin-stimulated adrenal glomerulosa cells. J Biol Chem 264:14078 19. Underwood RH, Greeley R, Clennon ET, Menachery AI, Braley LM, Williams GH 1988 Mass determination of polyphosphoinositides and inositol trisphosphate in rat adrenal glomerulosa cells with a microspectrophotometric method. Endocrinology 123:211 20. Isales CM, Bollag WB, Kiernan LC, Barrett PQ 1989 Effect of ANP on sustained aldosterone secretion stimulated by angiotensin II. Am J Physiol 256:C89 21. Hunyady L, Baukal AJ, Bor M, Ely JA, Catt KJ 1990 Regulation of 1,2-diacylglycerol production by angiotensin-II in bovine adrenal glomerulosa cells. Endocrinology 126:1001 22. Kojima I, Kojima K, Rasmussen H 1985 Role of calcium fluxes in the sustained phase of angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells. J Biol Chem 260:9177
1443
23. Barrett PQ, Kojima I, Kojima K, Zawalich K, Isales CM, Rasmussen H 1986 Short term memory in the calcium messenger system. Biochem J 238:905 24. Barrett PQ, Kojima I, Kojima K, Zawalich K, Isales CM, Rasmussen H 1986 Temporal patterns of protein phosphorylation after angiotensin II, A23187, and/or 12-O-tetradecanoyl phorbol-13-acetate in adrenal glomerulosa cells. Biochem J 238:893 25. Foster R, Lobo MV, Rasmussen H, Marusic E 1981 Calcium: its role in the mechanism of action of angiotensin II and potassium in aldosterone production. Endocrinology 109:2196 26. McCarthy R, Isales C, Bollag W, Rasmussen H, Barrett P 1989 Atrial natriuretic peptide differentially modulates T- and L-type calcium channels. Am J Physiol 258:F473 27. Bligh EG, Dyer WJ 1959 A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911 28. Preiss JE, Loomis CR, Bishop WR, Stein RB, Niedel JE, Bell RM 1986 Quantitative measurement of sn-l,2-diacylglycerols present in platelets, hepatocytes and ras- and sis-transformed normal rat kidney cells. J Biol Chem 261:8597 29. Eibl H, Kovatchev S 1981 Preparation of phospholipids and their analogs by phospholipase D. Methods Enzymol 72:632 30. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265 31. Lavie Y, Liscovitch M 1990 Activation of phospholipase D by sphingoid bases in NG108-15 neural-derived cells. J Biol Chem 265:3868 32. Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H 1989 Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev 10:496 33. Pagano RE, Longmuir KJ 1985 Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts. J Biol Chem 260:1909 34. Allan D, Thomas P, Michell RH 1978 Rapid transbilayer diffusion of 1,2-diacylglycerol and its relevance to control of membrane curvature. Nature 276:289 35. Ganong BR, Bell RM 1984 Transmembrane movement of phosphatidylglycerol and diacylglycerol sulfhydryl analogues. Biochemistry 23:4977 36. Nishizuka Y 1983 Calcium, phospholipid turnover and transmembrane signaling. Phil Trans R Soc Lond [B] 302:101 37. Lang U, Valloton MB 1987 Angiotensin II but not potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerulosa cells. J Biol Chem 262:8047 38. Ganz MB, Rasmussen L, Bollag WB, Rasmussen H 1990 Effect of buffer systems and pH; on the measurement of [Ca2+]i with fura 2. FASEB J 4:1638 39. Martin TW 1988 Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells. Biochim Biophys Acta 962:282
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 22:19 For personal use only. No other uses without permission. . All rights reserved.