73
Molectdar and Cellular Enakcrinolo~, 72 (19%) 73-80 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
02324
Key role of diacylglycerol-mediated 124ipoxygenase product formation in angiotensin II-induced aldosterone synthesis R. Natarajan, W.D. Dunn, N. Stern and 3. Nadler Section
ofEndocrinology, Administration
of Southern California Medical Center, Los Angeles, CA 90033, U.S.A., and Sepuiveda Center, University of California Los Angeles School of Medicine, Sepulveda, CA 91343, U.S.A.
Uniuersity
Veterans
(Received 13 March 1990; accepted 10 May 1990)
Key wor&: Diacyfglycerol; Angiotensin II; Aidosterone; IZHETE,
Arachidonic acid; Protein kinase C
We have shown earlier that the 1Zlipoxygenase product of arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (IZHETE), plays an important role in mediating angiotensin II {AII)-induced aldosterone secretion (J. Clin. Invest. (1987) 80, 1763). In the present study, we have evaluated whether diacylglycerol (DG) is the source of arachidonic acid giving rise to this 12-HETE. Treatment of rat adrenal glomerulosa cells with a DG lipase inhibitor, RHC 80267, which prevents conversion of DG to AA and HETEs, blocked AII-induced aldosterone and 12-HETE formation. In contrast, a DC kinase inhibitor, R59022, which prevents conversion of DG to phosphatidic acid, potentiated AII-induced aldosterone and 1ZHETE fo~ation. These two inhibitors block DG metabolism which would be expected to lead to increased DG levels and protein lcinase C activity and AII-induced steroidogenesis. However, only R59022 potentiated AII action while RHC 80267 was inhibitory. This suggests that conversion of DG to AA and 12-HETE is important for AI1 action. Further proof for this was obtained by measuring [3H]AA-labeled DG levels. The combination of the inhibitors significantly potentiated AII-induced DC formation even though this same combination was inhibitory on AII-induced aldosterone and 12-HETE. Thus, the inhibitory effect of RHC 80267 is due to blockade of AA release and not of DG formation. These results suggest that DG plays a dual role in AI1 action, both as an activator of protein kinase C and as a source of AA for 12-HETE formation.
Address for correspondence: Dr. Rama Natarajan, Division of Endocrinology, Univ. of Southern California Med. Ctr., 1200 N. State Street, Unit I, Room 18-632, Los Angeles, CA 90033. U.S.A. This work was supported by a grant from the NIH (ROlDK39721 (to J.N.)) and an American Heart Association, Los Angeles Affiliate Grant-in-Aid (to R.N.). J.N. is a recipient of a Clinical Investigator Award from the Heart, Lung, and Blood Institute (HL-01496). Presented in part at the 71st Annual Meeting of the Endocrine Society, Seattle, June 1989. 0303-7207/W/$03.50
Introduction In many cell types, hormones initiate their intracellular effects via the calcium messenger-phosphoinositide system (Berridge, 1984). Angiotensin II (AH), which is the major physiological regulator of aldosterone secretion in the adrenal, activates the phospholipase C-mediated hydrolysis of phosphoinositides to inositol n-&phosphate (IP,) and diacylglycerol (DG) (Kojima et al., 1984). The
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
74
importance of DG in cellular metabolism arises from the observation that protein kinase C activity is markedly augmented in the presence of DG (Nishizuka, 1984). Rasmussen and Barret (1984) and Kojima et al. (1984) have suggested that the cellular response to AI1 is obtained by the temporal integration of the IP3/calcium-calmodulin branch responsible for the initial transient response and the DG/protein kinase branch responsible for the sustained phase. DG thus plays a central role in AI1 action. DG can also be further metabolised by two pathways. In one pathway, DG is converted to phosphatidic acid by the enzyme DG kinase (Berridge and Irvine, 1984) and can thus reenter the phosphoinositide metabolism cycle. In the other pathway, arachidonic acid (AA) is released from DG by the action of DG lipase (Bell et al., 1979). AI1 action has also been shown to increase AA liberation from labeled calf adrenal glomerulosa cells (Kojima et al., 1985). AA in the adrenal can be metabolized to products of the cyclooxygenase and lipoxygenase (LO) pathways. Our recent studies in rat and human glomerulosa cells indicate that the 12-LO product, 12-hydroxyeicosatetraeonic acid (12-HETE) plays a key role in AII-induced aldosterone secretion (Nadler et al., 1987; Natarajan et al., 1988a). In the present study, we employed two specific inhibitors of DG metabolism, namely, RHC 80267 which blocks DG lipase (Sutherland and Amin, 1982) and R59022 which blocks DG kinase (DeChaffoy de Courcelles et al., 1985). We have examined the effect of these inhibitors individually and in combination on AII-induced stimulation of aldosterone and 12-HETE. In addition, we used a novel multistep mini-column procedure to assess the levels of DG during AI1 stimulation in the presence and absence of inhibitors of DG metabolism. We have employed these methods to determine the role of DG and its conversion to 1ZHETE in AI1 action. Materials and methods AI1 (human, synthetic) was obtained from Peninsula Laboratories (Belmont, CA, U.S.A.). The DG lipase inhibitor RHC 80267 was a gift from Rorer Central Research, Horsham, PA, U.S.A. It was initially dissolved in dimethyl
sulfoxide and subsequently diluted with water. The DG kinase inhibitor R59022 was obtained from Janssen Life Science Products, Olen, Belgium. It was initially dissolved in ethanol to a concentration of 2.5 X 10m2 M. This was then diluted IO-fold to 2.5 X 10e3 M with 0.005 N HCl followed by dilution with water to 10m3 M. 20 ~1 of this was added to 2.0 ml incubates to yield 10 5 M of R59022. Equal amounts of solvents were added to the control incubations. 12-HETE was obtained from Biomol Research Labs. (Philadelphia, PA, U.S.A.). 12-HETE was dried and resuspended in buffer by sonication.
Preparation and incubations of rat adrenal glomerulosa cells The cell supensions were freshly prepared using essentially the method of Braley and Williams (1977) as previously described (Nadler et al., 1987). In brief, adrenal capsules were removed from male Sprague-Dawley rats (200-225 g) and incubated in a modified Krebs-Ringer-bicarbonate buffer (KRBG) containing 1% bovine serum albumin (BSA; Pentex fraction V, Miles Scientific, Naperville, IL, U.S.A.), collagenase (3.7 mg/ml) (Worthington, Freehold, NJ, U.S.A.), deoxyribonuclease (0.05 mg/ml), glucose (2 mg/ml), L-glutamine (0.2 mg/ml), essential and non-essential amino acids (1.25% each, v/v) (Gibco, Grand Island, NY, U.S.A.). The washed cells were resuspended in the same buffer to yield about 100,000 cells/2 ml. The cell suspensions were preincubated for 10 min with the inhibitors prior to the addition of AIL At the end of the 1 h incubation period, a 0.3 ml aliquot was removed for aldosterone measurements while the remaining was stored at - 70” C for the measurement of 12-HETE. For experiments involving the measurement of DG, the cells were prelabeled with [3H]AA (1.75 pCi/ml, New England Nuclear Co., Boston, MA, U.S.A.) for 45 min at 37 o C in KRBG containing 0.02% BSA. The unesterified AA was removed by washing twice with KRBG containing 1.0% BSA. Washed cells were finally incubated in a volume of 2 ml of KRBG for various time intervals after addition of AI1 or the inhibitors. The reaction was terminated by the addition of 2 ml ice-cold methanol.
75
Measurement of aldosterone and 12-HETE Aldosterone was extracted from cell incubates with methylene chloride and measured by a specific radioimmunoassay (RIA) as described earlier (Nadler et al., 1987). 12-HETE was extracted using 500 mg Cl8 Bond Elut Mini Columns (Analytichem International, Torrance, CA, U.S.A.) and quantitated by a specific RIA as described earlier (Nadler et al., 1987; Natarajan et al., 1988b). The 12-HETE antiserum was obtained from Advanced Magnetics (Boston, MA, U.S.A.) and authentic [3H]12-HETE from New England Nuclear Co. Unlabeled 12-HETE standard was obtained from Biomol Research Laboratories (Philadelphia, PA, U.S.A.). The extracted 12HETE was further purified by our validated reverse-phase high-performance liquid chromatography (HPLC) method (Nadler et al., 1987). Extraction and measurements of DG The cell incubates containing 2 ml buffer and 2 ml methanol were subjected to total lipid extraction (Bligh and Dyer, 1959). 2 ml chloroform (CHCl,) was added to the mixture, vortexed and kept aside for 3 min. 2 ml more CHCl, was added, vortexed and centrifuged at 2000 rpm at 4’ C. The upper aqueous layer was reextracted with 4 ml CHCl,. The pooled lower organic layers were dried down and reconstituted in 0.5 ml CHCl,. This total lipid extract was then separated into various lipid classes using a novel multiple extraction procedure on 3 ml aminopropyl Bond Elut Columns with stainless steel frits (Analytichem International) by a modification of the method of Kaluzny et al. (1985). The total lipid extract in 0.5 ml CHCl, was loaded onto hexane (4 ml)-washed aminopropyl columns. The columns were eluted successively with 4 ml each solvent A (2 : 1 chloroform/isopropanol), solvent B (2% acetic acid in diethyl ether) and solvent C (methanol) to yield fractions I, II, and III respectively which contain the neutral lipids, fatty acids and phospholipids respectively. Fraction I (neutral lipids) was dried under nitrogen, reconstituted in 200 ~1 hexane and loaded on a new hexane-washed aminopropyl column. This was then eluted successively with 4 ml solvent D (hexane), 6 ml solvent E (1% diethyl ether, 10% methylene chloride in hexane), 12 ml solvent F (5% ethyl acetate in hexane), 4 ml solvent
G (50% ethyl acetate in hexane) and 4 ml solvent H (chloroform/methanol, 2 : 1) to yield fractions IV (cholesteryl esters), V (triglycerides), VI (cholesterol), VII (diglycerides) and VIII (monoglycerides) respectively. The percent radioactivity in fraction VII represents the percent of [3H]AAlabeled DG formed. Authentic cold diglyceride standards (1,2-diolein and 1,2-arachidonyl, stearoyl glycerol from Avanti Polar Lipids and Sigma Chemical Company, St. Louis, MO, U.S.A., respectively) and tritiated DG tracer ([3H]1,2arachidonyl, stearoyl glycerol (New England Nuclear Co.)) eluted over 90% in fraction VII. Percent efficiency of the complete extraction was 58 + 6. DG formation was further validated by normal phase HPLC using a 3 pm, 10 cm silica column (Perkin Elmer, Norwalk, CT, U.S.A.). Fraction VII from extraction was further purified on this HPLC column by using the isocratic solvent system of hexane/ isopropanol/ acetic acid (100 : 0.5 : 0.01, v/v/v) as described by Abe and Kogure (1986). The flow rate of the mobile phase was 2 ml/mm. Over 80% of radioactivity in fraction VII co-eluted with authentic cold and tritiated 1,2-DG
1 Radioactivity, PH] DG from cells Absorbance at 206nm. unlabeled DG standard
.:
-E
I
R
‘F .-
1 \
I,\____-___JY&_ J f
xc-5 ;
4 & ;
Fig. 1. Normal phase HPLC separation of labeled 1,2-DG from rat adrenal glomerulosa cells. Fraction 7 obtained from the multistage lipid extraction on amino-propyl columns was dried and reconstituted in the mobile phase (n-hexane/ isopropanol/acetic acid (100 : 0.5 : 0.01, v/v/v)) before injection. Cold 1,2-DG standard shown is 14 pg of 1,2-arachidonyl, stearoyl glycerol (absorbance at 206 nm). The corresponding tritiatcd 1,2-DG standard also comigrated with the cold standard. The flow rate was 2 ml/mm.
76
(Fig. 1) confirming fraction VII as 1,2-DG. Retention time of 1,2-DG standards in this solvent system was 12.9 min. There was some isomerization of 1,2-DG to 1,3-DG noted (retention time 7.6 mm). The cold standards (14 pg) were detected by UV absorption at 206 nm. Data analysis All results are Analysis of variance with experimental sons Duncan’s test was performed on (GCRC RR-43).
expressed as the mean + SE. was used to compare control values. For multiple compariwas also used. Data analysis a CLINFO computer system
Results
The effect of inhibitors of DG metabolism on AII-induced aldosterone synthesis Fig. 2 shows the effect of the inhibitors RHC 80267 and R59022 on AI1 stimulation of aldosterone. R59022 was used at 10e5 M, the dose shown to effectively and specifically block DG kinase. RHC 80267 was employed at both lo-’ M and 10P6 M. RHC 80267 at lo-’ M and R59022 at 10P5 M did not alter basal aldosterone levels. RHC 80267 at 10e6 M had a slight inhibitory effect on basal aldosterone (basal, 3.5 & 0.3 ng vs. RHC 80267, 2.9 t_ 0.3 ng). Fig. 2 shows the contrasting effects of these two inhibitors on AII-induced aldosterone synthesis. The DG lipase inhibitor RHC 80267 (lo-’ M) caused a significant attenuation of AII-induced aldosterone synthesis (basal, 3.5 f 0.3 ng/106 cells/h; AII, 23.4 k 1.2 ng, p < 0.001 vs. basal; AI1 + RHC 80267, 11.4 + 1.1 ng, p < 0.001 vs. AII). In contrast, the DG kinase inhibitor R59022 (lop5 M) caused a marked potentiation of the AI1 effect (AI1 + R59022, 57.8 + 9.4 ng, p < 0.02 vs. AII). Further, the effect of R59022 was blocked by the lipase inhibitor RHC 80267 (AI1 + RHC 80267 + R59022, 9.7 + 2.1 ng, p -=z0.01 vs. AI1 + R59022). The effect aldosterone K+ at dosterone cells/h ( p
of DG-lipase inhibition on K +-induced stimulation 10.7 mEq concentration stimulated alsynthesis 10.5 + 3 to 67 +Z25 ng/106 < 0.04, n = 9). In addition, RHC 80267
1 :.I... ::: ::: :::: :::: :::: :::: :::: ::: ::::
BASAL
Alll10-%4
All+ RHCB0267 (10.‘M)
All + R59022 (lm.4)
All + R59022+ RHC 90267
Fig. 2. The effect of inhibitors of DG metabolism on AII-induced aldosterone synthesis. The results are expressed as mean *SE from 4-6 separate experiments performed in triplicate. *p -C0.001 vs. basal; * * p < 0.01 vs. AII; * * * p -C0.02 vs. AH.
at lo-’ M inhibited K+-mediated aldosterone stimulation (22 -t_6 ng/106 cells/h, p < 0.01). The effect of inhibitors of DG metabolism on AII-induced I2-HETE formation AI1 (10e9 M) caused stimulation of 12-HETE levels (Fig. 3). As with effects on aldosterone, the two inhibitors of DG metabolism had contrasting effects on 12-HETE formation. RHC 80267, at the same dose that decreased AII-induced aldosterone levels, also significantly blocked AII-mediated stimulation of 12-HETE levels (Fig. 3) (basal, 6.9 + 0.3 ng/106 cells/h; AII, 15.5 k 2.5 ng, p < 0.05 vs. basal; AI1 + RHC 80267 8.6 + 0.8 ng, p < 0.02 vs. AII). In contrast, R59022 significantly potentiated AI1 action (AI1 + R59022, 23.2 f 2.2
77 TABLE
1
EFFECT OF 12-HETE ON RESTORING ALDOSTERONE SYNTHESIS DURING HIBITION Results
are expressed
AII-INDUCED DG LIPASE IN-
as mean f SE (n = 9).
Treatment
Aldosterone (ng/106 cells/h)
Basal AI1 (10K9 M) AI1 + RHC 80267 (lo-’ M) AI1 + RHC 80267 + I2-HETE (10K6 M)
5.3 f 0.4 29.5 f 1.0
< 0.01 vs. basal
18.5+1.2
i 0.01 vs. AI1
28.4k 1.7
i 0.01 vs. AI1 + RHC 80267 and NS vs. AI1
P
lated DG in two distinct phases in these rat adrenal glomerulosa cells. An early peak occurred at 30 s (139 _t 12% control) while a late peak occurred at 10 min (137 -I_4% control). BASAL
All110%41
A;;2\vC (10-W
All+ All+ R59022+ R59022 (lo-5b.4) AH030267
Fig. 3. The effect of inhibitors of DG metabolism on AII-induced 12-HETE levels. Values are mean * SE from four experiments performed in triplicate. * p < 0.05 vs. basal; * * p < 0.02 vs. AH; * * * p < 0.02 vs. AH.
ng, p < 0.02 vs. AII). Similar to its effects on aldosterone, RHC 80267 blocked the potentiating effect of R59022 bringing 1ZHETE levels back to control (AI1 + RHC 80267 + R59022,7.5 + 0.8 ng, p < 0.01 vs. AI1 + R59022). The effect of 12-HETE addition on AII-induced aldosterone during DG Iipase inhibition Table 1 depicts the effect of 12-HETE addition on AII-induced aldosterone synthesis during DG lipase inhibition. AII-induced action during inhibition by RHC 80267 is completely restored by the addition of the 12-LO product 12-HETE (Table 1). The effect of AII on DG levels Fig. 4 depicts the time course of AII-induced DG levels in glomerulosa cells labeled with [3H]AA. We observed that AI1 (10e9 M) stimu-
The effect of AII and the inhibitors of DG metabolism on DG levels Fig. 5 shows the effect of AI1 alone and with inhibitors of DG metabolism on [3H]AA-labeled DG levels. Results shown are from 10 min experi-
* ** l/y -
I
H
Control
Au10-9M
I
J
A
TlME(MIN.)
Fig. 4. Time course of AII-stimulated diacylglycerol formation. The results are percent control radioactivity in DG fraction and are expressed as meanf SE from 3-5 experiments performed in duplicate or triplicate. * p < 0.04 vs. control; **p < 0.01 vs. control. Incubation and lipid extractions were carried out as described in Materials and Methods.
78
5
1707
c Y z
160-
x z
150-
f _ 5 F
140
Y
d J 0 E 6
120
110
0 8 2
100
Lz 130 : ! AIIIlO-WI
All+ RHC80267
All + I359022
All + R59022+ RHC80267
Fig. 5. The effect of AI1 alone and with inhibitors of DG metabolism on [3H]DG levels. Results shown are data obtained from 10 min incubations. Values are mean k SE from 3-5 experiments performed in duplicate or triplicate. *p < 0.01. RHC 80267 and R59022 were used at lo-’ M and 10m5 M resp.
ments. All stimulated DG levels to 137 k 4% of basal ( p < 0.001). RHC 80267 used alone did not significantly alter the effect of All on DG levels (All + RHC 80267,131 + 3%, p < 0.001 vs. basal). However, R59022 had a slight potentiating effect vs. All alone (All + R59022, 148 + 5% basal). In contrast as seen in Fig. 5 the combination of the two inhibitors significantly potentiated the All effect on stimulation of DG (167 + 6% basal, p < 0.01 vs. All). Discussion
DG plays an important role in the signal transduction of All action in the adrenal glomerulosa cell. DG can directly activate protein kinase C. However, DG can also be converted to phosphatidic acid by DG kinase or lead to AA release via DG lipase. In a preliminary study, a DG lipase inhibitor reduced All-induced aldosterone synthesis in human glomerulosa cells suggesting that AA release from DG may also be important in All action (Natarajan et al., 1988b).
In the present report we found that All stimulated DG levels in a biphasic fashion with peaks at 30 s and 10 min. These are the first results of DG levels in rat glomerulosa cells and are similar to those reported earlier in bovine cells (Kojima et al., 1984; Hunyady et al., 1990). The mechanism and role of this biphasic DG response after All stimulation are not clear. However, previous studies suggest that this may be due to a shift in substrate requirement from phosphatidylinositol biphosphate to phosphatidylinositol (Griendling et al., 1986). Sutherland and Amin (1982) first described RHC 80267 as a potent and selective inhibitor of canine platelet DG-lipase. In more recent studies RHC 80267 has been used to evaluate the role of AA release in DG action in endocrine tissues. Several reports have shown that RHC 80267 can inhibit prolactin release (Canonico et al., 1985; Camoratto and Grandison, 1987; Judd et al., 1988). Chang et al. (1988) showed that DG metabolism via DG lipase is an important step in gonadotropin-releasing hormone-induced stimulation of luteinizing and follicle-stimulating hormone secretion. In the latter study, RHC 80267 action was specific since it did not alter phospholipase A, or C, protein kinase C or Ca*+ responses in cells. In our earlier studies with human adrenal cells, RHC 80267 action at 5 PM was specific to All and Kf-induced aldosterone synthesis and did not alter ACTH effects (Natarajan et al., 1988b). In the current study, RHC 80267 synthesis was effective at concentrations as low as lo-’ M in blocking All-induced aldosterone and 12-HETE. We have also shown that this dose of RHC 80267 inhibits AII-induced release of [‘H]AA from prelabeled rat adrenal glomerulosa cells (unpublished observations). RHC 80267 at lo-’ M concentration also inhibited K+-induced aldosterone synthesis. These results are similar to our previous studies in human adrenal cells (Natarajan et al., 1988b). Preliminary studies also suggest that K+ induces [3H]AA release and that K+-induced aldosterone synthesis is markedly potentiated in rat adrenal cells incubated in the presence of 1 PM arachidonic acid (unpublished observations). Our earlier studies have shown that K+ does not induce 12-HETE formation (Nadler et al., 1987). Therefore, AA
79
itself or other non-LO metabolites of AA may play an important role in Kf-mediated aldosterone synthesis. Additional studies will be required to evaluate these effects. In the current study we did not evaluate the effect of RHC 80267 on ACTH action. We previously showed that in human adrenal cells RHC 80267 did not alter ACTH-induced effects (Natarajan et al., 1988b). However, very recent evidence (Cozza et al., 1990a, b) suggests that ACTH can induce the de novo synthesis of DG in bovine adrenal cells, suggesting that there may be species variation in ACTH action. Therefore, additional studies will be required to evaluate whether in the rat, ACTH action involves DG formation and AA release. R59022 has been reported to be a selective inhibitor of DG kinase in both intact human platelets and human red cell membranes (DeChaffoy de Courcelles et al., 1985). In thrombinactivated platelets, addition of R59022 resulted in a marked elevation of DG levels, decreased formation of phosphatidic acid and increased protein kinase C activity. The IC,, for DG kinase inhibition was 3.8 X lop6 M and approximately 80% inhibition was obtained at 10e5 M. Most studies with R59022 have employed 10m5 M concentration. R59022 has been used by several workers as a valuable tool for the amplification of DG signals in studies related to mechanisms of signal transduction. Muid et al. (1987) employed R59022 to determine the important role of protein kinase C and arachidonate in the signal transduction of the respiratory burst in the neutrophil. It has also been useful in elucidating the contribution of the protein kinase C pathway in long-term mitogenesis (Morris et al., 1988). In the present study, the DG kinase inhibitor, R59022, caused a marked potentiation of the AI1 effects on aldosterone and 12-HETE. RHC 80267 and R59022 are both inhibitors of DG metabolism, a process that should lead to build up of DG which in turn should lead to increased protein kinase C activity and AI1 action. However, only R59022 potentiated AI1 action whereas RHC 80267 was inhibitory. We hypothesized that RHC 80267 was blocking AI1 action due to its inhibitory effect on AA and 12-HETE formation. In order to test this, we examined the effect of these
inhibitors on AII-induced 12-HETE formation. Similar to its effect on aldosterone, RHC 80267 also blocked AII-induced 12-HETE levels, while R59022 caused a marked potentiation of the 12-LO product. Furthermore, RHC 80267 also prevented the potentiating effect of R59022 on 12-HETE formation. 12-HETE addition could restore the effects of AI1 during DG lipase inhibition, thus supporting the specificity of action of RHC 80267. These results are consistent with the hypothesis that 12-HETE formation from DG is an important step in AI1 action in the adrenal. Therefore, increasing DG levels potentiates AII-mediated aldosterone synthesis only when DG can form AA products via DG lipase. We measured [‘H]DG levels in the presence of AI1 and these inhibitors in order to provide additional support for the action of these agents. Neither the inhibitors RHC 80267 nor R59022 significantly altered AII-induced stimulation of DG at 10 min. Moreover, the combination of the two inhibitors significantly potentiated AI1 effect on DG even though this same combination was inhibitory on AII-induced aldosterone and 12HETE. Thus the inhibitory effect of RHC 80267 on AII-induced aldosterone and 12-HETE levels was not due to a blockade of DG formation, but instead due to a blockade of AA release. The major sources of AA in cells are from phosphatidylcholine, phosphatidylethanolamine or phosphatidic acid by the action of phospholipase A, (Bills et al., 1977; Lapetina et al., 1981) or from phosphoinositides by the sequential action of phospholipase C and DG lipase (Bell et al., 1979; Chau and Tai, 1981). Previous studies have shown that in the adrenal, AI1 does not activate phospholipase A, (Hunyady et al., 1982). Thus, DG via DG lipase could provide an important source of AA and 12-HETE in adrenal glomerulosa cells. Our earlier studies have shown the importance of 1ZHETE in AII-mediated aldosterone synthesis in rat and human adrenal glomerulosa cells (Nadler et al., 1987; Natarajan et al., 1988a). The current study confirms that the origin of 12-HETE is from AA derived from DG. We have proposed that AA metabolites of the 12-LO pathway could facilitate the action of protein kinase C in mediating AI1 action. The possible mechanisms by which 12-HETE could achieve
80
this is by the activation of protein kinase C directly or by the activation of Ca2+ or both. Several fatty acids including HETEs have been shown to activate protein kinase C (Murakami and Routtenberg, 1985; Hansson et al., 1986) and recently AA and 12-HETE have been shown to activate the r-subspecies of protein kinase C directly in a Ca’+-independent manner (Shearman et al., 1989). Seifert et al. (1988) have suggested that fatty acids and DG may be synergistically involved in hormonal stimulation of protein kinase C. We have also shown using fura- Ca*+ measurements in rat cells that 12-HETE also stimulates Ca*+ in a manner similar to AI1 (Stern et al., 1988). Further studies will be needed to fully evaluate the role of protein kinase C and Ca2+ release by 12-HETE in mediating AI1 action in the adrenal. In summary, this study suggests that DG generated by receptor activation plays a central role in AI1 action, not only as an endogenous activator of protein kinase C, but also as a precursor of active lipoxygenase metabolites of AA that mediate AI1 action. Acknowledgement The authors wish to thank Ms. Jenny Yang for her secretarial assistance in typing the manuscript. References Abe, K. and Kogure, K. (1986) J. Neurochem. 47, 577-582. Bell, R.L., Kennerly, D.A., Stanford, N. and Majerus, P.W. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3238-3241. Berridge, M.J. (1984) B&hem. J. 220, 345-360. Berridge, M.J. and Irvine, R.F. (1984) Nature 312, 315-321. Bills, T.K., Smith, J.B. and Silver, M.J. (1977) J. Clin. Invest. 60, 1-6. B&h, E.G. and Dyer, W.J. (1959) Can. J. B&hem. Physiol. 37, 911-917. Braley, L.M. and Williams, G.H. (1977) Am. J. Physiol. 233, 402-406. Camoratto, A.M. and Grandison, L. (1987) Life Sci. 40, 275281. Canonico, P.L., Cronin, M.J. and Macleod, R.M. (1985) Life Sci. 36, 997-1002. Chang, J.P., Morgan, R.O. and Catt, K.J. (1988) J. Biol. Chem. 263. 18614-18620.
Chau, L.Y. and Tai, H.H. (1981) B&hem. Biophys. Res. Commun. 100, 168881695. Cozza, E.N., Vila, C.M.D., Acevedo-Duncan, M., GomezSanchez, C.E. and Farese, R.V. (1990a) J. Steroid B&hem. 35, 343-351. Cozza, E.N., Vila, C.M.D., Acevedo-Duncan, M., Farese, V. and Gomez-Sanchez, E.C. (1990b) Endocrinology 126, 2169-2176. DeChaffoy de Courcelles, D., Roevens, P. and Van Belle, H. (1985) J. Biol. Chem. 260, 15762-15770. Griendling, K.K., Rittenhouse, SE., Brock, T.A., Ekstein, L.S., Gimbrone, M.A. and Alexander, W.R. (1986) J. Biol. Chem. 251, 5901-5906. Hansson, A., Serhan, C.N., Haeggstrom, J., Sunberg, M.I. and Samuelsson, B. (1986) Biochem. Biophys. Res. Commun. 134,1215-1222. Hunyday, L., Balla, T., Enyedi, P. and Spat, A. (1985) Biothem. Pharmacol. 34, 3439-3444. Hunyday, L., Baukal, A.J.. Bor, M., Ely, J.A. and Catt, K.J. (1990) Endocrinology 126, 100-1008. Judd, A.M., Ross, P.C., Spangelo, B.L. and Macleod, R.M. (1988) Mol. Cell. Endocrinol. 57, 115-121. Kaluzny, M.A., Duncan, L.A., Merrit, M.V. and Epps, D.E. (1985) J. Lipid Res. 26, 1355144. Kojima, I., Kojima, K., Kreulter, D. and Rasmussen, H. (1984) J. Biol. Chem. 259, 14448814457. Kojima, I., Kojima. K. and Rasmussen, H. (1985) Endocrinology 117, 1057-1066. Lapetina, E.J., Billah, M.M. and Cuatracasas, P. (1981) J. Biol. Chem. 256, 5037-5040. Morris, C., Rice, P. and Rozeugurt, E. (1988) Biochem. Biophys. Res. Commun. 155, 561-568. Muid, R.E., Penfield, A. and Dale, M.M. (1987) Biochem. Biophys. Res. Commun. 143, 630-637. Murakami. K. and Routtenberg, A. (1985) FEBS Lett. 192, 189-193. Nadler, J.L., Natarajan, R. and Stern, N. (1987) J. Clin. Invest. 80, 176331769. Natarajan, R., Stem, N., Hsueh. W., Do, Y. and Nadler, J. (1988a) J. Clin. Endocrinol Metab. 67, 584-591. Natarajan, R.. Stern, N. and Nadler, J. (1988b) Biochem. Biophys. Res. Commun. 156, 717-724. Nishizuka, Y. (1984) Nature 308, 693-698. Rasmussen, H. and Barret, P.Q. (1984) Physiol. Rev. 64, 938984. Seifert, R., Schachtele, C., Rosenthal, W. and Schultz, G. (1988) B&hem. Biophys. Res. Commun. 154, 20-26. Shearman, M., Naor, Z., Sekiguchi, K., Kishimoto, A. and Nishizuka, Y. (1989) FEBS Lett. 243, 1777182. Stem, N., Yanagawa, N., Natarajan, R., Tuck, M.L. and Nadler, J. (1988) 70th Annual Meeting of the Endocrine Society, New Orleans, No. 1141. Sutherland, C.A. and Amin. D. (1982) J. Biol. Chem. 257, 14006-14010.
Molectdar and Cellular Enakcrinolo~, 72 (19%) 73-80 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
02324
Key role of diacylglycerol-mediated 124ipoxygenase product formation in angiotensin II-induced aldosterone synthesis R. Natarajan, W.D. Dunn, N. Stern and 3. Nadler Section
ofEndocrinology, Administration
of Southern California Medical Center, Los Angeles, CA 90033, U.S.A., and Sepuiveda Center, University of California Los Angeles School of Medicine, Sepulveda, CA 91343, U.S.A.
Uniuersity
Veterans
(Received 13 March 1990; accepted 10 May 1990)
Key wor&: Diacyfglycerol; Angiotensin II; Aidosterone; IZHETE,
Arachidonic acid; Protein kinase C
We have shown earlier that the 1Zlipoxygenase product of arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (IZHETE), plays an important role in mediating angiotensin II {AII)-induced aldosterone secretion (J. Clin. Invest. (1987) 80, 1763). In the present study, we have evaluated whether diacylglycerol (DG) is the source of arachidonic acid giving rise to this 12-HETE. Treatment of rat adrenal glomerulosa cells with a DG lipase inhibitor, RHC 80267, which prevents conversion of DG to AA and HETEs, blocked AII-induced aldosterone and 12-HETE formation. In contrast, a DC kinase inhibitor, R59022, which prevents conversion of DG to phosphatidic acid, potentiated AII-induced aldosterone and 1ZHETE fo~ation. These two inhibitors block DG metabolism which would be expected to lead to increased DG levels and protein lcinase C activity and AII-induced steroidogenesis. However, only R59022 potentiated AII action while RHC 80267 was inhibitory. This suggests that conversion of DG to AA and 12-HETE is important for AI1 action. Further proof for this was obtained by measuring [3H]AA-labeled DG levels. The combination of the inhibitors significantly potentiated AII-induced DC formation even though this same combination was inhibitory on AII-induced aldosterone and 12-HETE. Thus, the inhibitory effect of RHC 80267 is due to blockade of AA release and not of DG formation. These results suggest that DG plays a dual role in AI1 action, both as an activator of protein kinase C and as a source of AA for 12-HETE formation.
Address for correspondence: Dr. Rama Natarajan, Division of Endocrinology, Univ. of Southern California Med. Ctr., 1200 N. State Street, Unit I, Room 18-632, Los Angeles, CA 90033. U.S.A. This work was supported by a grant from the NIH (ROlDK39721 (to J.N.)) and an American Heart Association, Los Angeles Affiliate Grant-in-Aid (to R.N.). J.N. is a recipient of a Clinical Investigator Award from the Heart, Lung, and Blood Institute (HL-01496). Presented in part at the 71st Annual Meeting of the Endocrine Society, Seattle, June 1989. 0303-7207/W/$03.50
Introduction In many cell types, hormones initiate their intracellular effects via the calcium messenger-phosphoinositide system (Berridge, 1984). Angiotensin II (AH), which is the major physiological regulator of aldosterone secretion in the adrenal, activates the phospholipase C-mediated hydrolysis of phosphoinositides to inositol n-&phosphate (IP,) and diacylglycerol (DG) (Kojima et al., 1984). The
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
74
importance of DG in cellular metabolism arises from the observation that protein kinase C activity is markedly augmented in the presence of DG (Nishizuka, 1984). Rasmussen and Barret (1984) and Kojima et al. (1984) have suggested that the cellular response to AI1 is obtained by the temporal integration of the IP3/calcium-calmodulin branch responsible for the initial transient response and the DG/protein kinase branch responsible for the sustained phase. DG thus plays a central role in AI1 action. DG can also be further metabolised by two pathways. In one pathway, DG is converted to phosphatidic acid by the enzyme DG kinase (Berridge and Irvine, 1984) and can thus reenter the phosphoinositide metabolism cycle. In the other pathway, arachidonic acid (AA) is released from DG by the action of DG lipase (Bell et al., 1979). AI1 action has also been shown to increase AA liberation from labeled calf adrenal glomerulosa cells (Kojima et al., 1985). AA in the adrenal can be metabolized to products of the cyclooxygenase and lipoxygenase (LO) pathways. Our recent studies in rat and human glomerulosa cells indicate that the 12-LO product, 12-hydroxyeicosatetraeonic acid (12-HETE) plays a key role in AII-induced aldosterone secretion (Nadler et al., 1987; Natarajan et al., 1988a). In the present study, we employed two specific inhibitors of DG metabolism, namely, RHC 80267 which blocks DG lipase (Sutherland and Amin, 1982) and R59022 which blocks DG kinase (DeChaffoy de Courcelles et al., 1985). We have examined the effect of these inhibitors individually and in combination on AII-induced stimulation of aldosterone and 12-HETE. In addition, we used a novel multistep mini-column procedure to assess the levels of DG during AI1 stimulation in the presence and absence of inhibitors of DG metabolism. We have employed these methods to determine the role of DG and its conversion to 1ZHETE in AI1 action. Materials and methods AI1 (human, synthetic) was obtained from Peninsula Laboratories (Belmont, CA, U.S.A.). The DG lipase inhibitor RHC 80267 was a gift from Rorer Central Research, Horsham, PA, U.S.A. It was initially dissolved in dimethyl
sulfoxide and subsequently diluted with water. The DG kinase inhibitor R59022 was obtained from Janssen Life Science Products, Olen, Belgium. It was initially dissolved in ethanol to a concentration of 2.5 X 10m2 M. This was then diluted IO-fold to 2.5 X 10e3 M with 0.005 N HCl followed by dilution with water to 10m3 M. 20 ~1 of this was added to 2.0 ml incubates to yield 10 5 M of R59022. Equal amounts of solvents were added to the control incubations. 12-HETE was obtained from Biomol Research Labs. (Philadelphia, PA, U.S.A.). 12-HETE was dried and resuspended in buffer by sonication.
Preparation and incubations of rat adrenal glomerulosa cells The cell supensions were freshly prepared using essentially the method of Braley and Williams (1977) as previously described (Nadler et al., 1987). In brief, adrenal capsules were removed from male Sprague-Dawley rats (200-225 g) and incubated in a modified Krebs-Ringer-bicarbonate buffer (KRBG) containing 1% bovine serum albumin (BSA; Pentex fraction V, Miles Scientific, Naperville, IL, U.S.A.), collagenase (3.7 mg/ml) (Worthington, Freehold, NJ, U.S.A.), deoxyribonuclease (0.05 mg/ml), glucose (2 mg/ml), L-glutamine (0.2 mg/ml), essential and non-essential amino acids (1.25% each, v/v) (Gibco, Grand Island, NY, U.S.A.). The washed cells were resuspended in the same buffer to yield about 100,000 cells/2 ml. The cell suspensions were preincubated for 10 min with the inhibitors prior to the addition of AIL At the end of the 1 h incubation period, a 0.3 ml aliquot was removed for aldosterone measurements while the remaining was stored at - 70” C for the measurement of 12-HETE. For experiments involving the measurement of DG, the cells were prelabeled with [3H]AA (1.75 pCi/ml, New England Nuclear Co., Boston, MA, U.S.A.) for 45 min at 37 o C in KRBG containing 0.02% BSA. The unesterified AA was removed by washing twice with KRBG containing 1.0% BSA. Washed cells were finally incubated in a volume of 2 ml of KRBG for various time intervals after addition of AI1 or the inhibitors. The reaction was terminated by the addition of 2 ml ice-cold methanol.
75
Measurement of aldosterone and 12-HETE Aldosterone was extracted from cell incubates with methylene chloride and measured by a specific radioimmunoassay (RIA) as described earlier (Nadler et al., 1987). 12-HETE was extracted using 500 mg Cl8 Bond Elut Mini Columns (Analytichem International, Torrance, CA, U.S.A.) and quantitated by a specific RIA as described earlier (Nadler et al., 1987; Natarajan et al., 1988b). The 12-HETE antiserum was obtained from Advanced Magnetics (Boston, MA, U.S.A.) and authentic [3H]12-HETE from New England Nuclear Co. Unlabeled 12-HETE standard was obtained from Biomol Research Laboratories (Philadelphia, PA, U.S.A.). The extracted 12HETE was further purified by our validated reverse-phase high-performance liquid chromatography (HPLC) method (Nadler et al., 1987). Extraction and measurements of DG The cell incubates containing 2 ml buffer and 2 ml methanol were subjected to total lipid extraction (Bligh and Dyer, 1959). 2 ml chloroform (CHCl,) was added to the mixture, vortexed and kept aside for 3 min. 2 ml more CHCl, was added, vortexed and centrifuged at 2000 rpm at 4’ C. The upper aqueous layer was reextracted with 4 ml CHCl,. The pooled lower organic layers were dried down and reconstituted in 0.5 ml CHCl,. This total lipid extract was then separated into various lipid classes using a novel multiple extraction procedure on 3 ml aminopropyl Bond Elut Columns with stainless steel frits (Analytichem International) by a modification of the method of Kaluzny et al. (1985). The total lipid extract in 0.5 ml CHCl, was loaded onto hexane (4 ml)-washed aminopropyl columns. The columns were eluted successively with 4 ml each solvent A (2 : 1 chloroform/isopropanol), solvent B (2% acetic acid in diethyl ether) and solvent C (methanol) to yield fractions I, II, and III respectively which contain the neutral lipids, fatty acids and phospholipids respectively. Fraction I (neutral lipids) was dried under nitrogen, reconstituted in 200 ~1 hexane and loaded on a new hexane-washed aminopropyl column. This was then eluted successively with 4 ml solvent D (hexane), 6 ml solvent E (1% diethyl ether, 10% methylene chloride in hexane), 12 ml solvent F (5% ethyl acetate in hexane), 4 ml solvent
G (50% ethyl acetate in hexane) and 4 ml solvent H (chloroform/methanol, 2 : 1) to yield fractions IV (cholesteryl esters), V (triglycerides), VI (cholesterol), VII (diglycerides) and VIII (monoglycerides) respectively. The percent radioactivity in fraction VII represents the percent of [3H]AAlabeled DG formed. Authentic cold diglyceride standards (1,2-diolein and 1,2-arachidonyl, stearoyl glycerol from Avanti Polar Lipids and Sigma Chemical Company, St. Louis, MO, U.S.A., respectively) and tritiated DG tracer ([3H]1,2arachidonyl, stearoyl glycerol (New England Nuclear Co.)) eluted over 90% in fraction VII. Percent efficiency of the complete extraction was 58 + 6. DG formation was further validated by normal phase HPLC using a 3 pm, 10 cm silica column (Perkin Elmer, Norwalk, CT, U.S.A.). Fraction VII from extraction was further purified on this HPLC column by using the isocratic solvent system of hexane/ isopropanol/ acetic acid (100 : 0.5 : 0.01, v/v/v) as described by Abe and Kogure (1986). The flow rate of the mobile phase was 2 ml/mm. Over 80% of radioactivity in fraction VII co-eluted with authentic cold and tritiated 1,2-DG
1 Radioactivity, PH] DG from cells Absorbance at 206nm. unlabeled DG standard
.:
-E
I
R
‘F .-
1 \
I,\____-___JY&_ J f
xc-5 ;
4 & ;
Fig. 1. Normal phase HPLC separation of labeled 1,2-DG from rat adrenal glomerulosa cells. Fraction 7 obtained from the multistage lipid extraction on amino-propyl columns was dried and reconstituted in the mobile phase (n-hexane/ isopropanol/acetic acid (100 : 0.5 : 0.01, v/v/v)) before injection. Cold 1,2-DG standard shown is 14 pg of 1,2-arachidonyl, stearoyl glycerol (absorbance at 206 nm). The corresponding tritiatcd 1,2-DG standard also comigrated with the cold standard. The flow rate was 2 ml/mm.
76
(Fig. 1) confirming fraction VII as 1,2-DG. Retention time of 1,2-DG standards in this solvent system was 12.9 min. There was some isomerization of 1,2-DG to 1,3-DG noted (retention time 7.6 mm). The cold standards (14 pg) were detected by UV absorption at 206 nm. Data analysis All results are Analysis of variance with experimental sons Duncan’s test was performed on (GCRC RR-43).
expressed as the mean + SE. was used to compare control values. For multiple compariwas also used. Data analysis a CLINFO computer system
Results
The effect of inhibitors of DG metabolism on AII-induced aldosterone synthesis Fig. 2 shows the effect of the inhibitors RHC 80267 and R59022 on AI1 stimulation of aldosterone. R59022 was used at 10e5 M, the dose shown to effectively and specifically block DG kinase. RHC 80267 was employed at both lo-’ M and 10P6 M. RHC 80267 at lo-’ M and R59022 at 10P5 M did not alter basal aldosterone levels. RHC 80267 at 10e6 M had a slight inhibitory effect on basal aldosterone (basal, 3.5 & 0.3 ng vs. RHC 80267, 2.9 t_ 0.3 ng). Fig. 2 shows the contrasting effects of these two inhibitors on AII-induced aldosterone synthesis. The DG lipase inhibitor RHC 80267 (lo-’ M) caused a significant attenuation of AII-induced aldosterone synthesis (basal, 3.5 f 0.3 ng/106 cells/h; AII, 23.4 k 1.2 ng, p < 0.001 vs. basal; AI1 + RHC 80267, 11.4 + 1.1 ng, p < 0.001 vs. AII). In contrast, the DG kinase inhibitor R59022 (lop5 M) caused a marked potentiation of the AI1 effect (AI1 + R59022, 57.8 + 9.4 ng, p < 0.02 vs. AII). Further, the effect of R59022 was blocked by the lipase inhibitor RHC 80267 (AI1 + RHC 80267 + R59022, 9.7 + 2.1 ng, p -=z0.01 vs. AI1 + R59022). The effect aldosterone K+ at dosterone cells/h ( p
of DG-lipase inhibition on K +-induced stimulation 10.7 mEq concentration stimulated alsynthesis 10.5 + 3 to 67 +Z25 ng/106 < 0.04, n = 9). In addition, RHC 80267
1 :.I... ::: ::: :::: :::: :::: :::: :::: ::: ::::
BASAL
Alll10-%4
All+ RHCB0267 (10.‘M)
All + R59022 (lm.4)
All + R59022+ RHC 90267
Fig. 2. The effect of inhibitors of DG metabolism on AII-induced aldosterone synthesis. The results are expressed as mean *SE from 4-6 separate experiments performed in triplicate. *p -C0.001 vs. basal; * * p < 0.01 vs. AII; * * * p -C0.02 vs. AH.
at lo-’ M inhibited K+-mediated aldosterone stimulation (22 -t_6 ng/106 cells/h, p < 0.01). The effect of inhibitors of DG metabolism on AII-induced I2-HETE formation AI1 (10e9 M) caused stimulation of 12-HETE levels (Fig. 3). As with effects on aldosterone, the two inhibitors of DG metabolism had contrasting effects on 12-HETE formation. RHC 80267, at the same dose that decreased AII-induced aldosterone levels, also significantly blocked AII-mediated stimulation of 12-HETE levels (Fig. 3) (basal, 6.9 + 0.3 ng/106 cells/h; AII, 15.5 k 2.5 ng, p < 0.05 vs. basal; AI1 + RHC 80267 8.6 + 0.8 ng, p < 0.02 vs. AII). In contrast, R59022 significantly potentiated AI1 action (AI1 + R59022, 23.2 f 2.2
77 TABLE
1
EFFECT OF 12-HETE ON RESTORING ALDOSTERONE SYNTHESIS DURING HIBITION Results
are expressed
AII-INDUCED DG LIPASE IN-
as mean f SE (n = 9).
Treatment
Aldosterone (ng/106 cells/h)
Basal AI1 (10K9 M) AI1 + RHC 80267 (lo-’ M) AI1 + RHC 80267 + I2-HETE (10K6 M)
5.3 f 0.4 29.5 f 1.0
< 0.01 vs. basal
18.5+1.2
i 0.01 vs. AI1
28.4k 1.7
i 0.01 vs. AI1 + RHC 80267 and NS vs. AI1
P
lated DG in two distinct phases in these rat adrenal glomerulosa cells. An early peak occurred at 30 s (139 _t 12% control) while a late peak occurred at 10 min (137 -I_4% control). BASAL
All110%41
A;;2\vC (10-W
All+ All+ R59022+ R59022 (lo-5b.4) AH030267
Fig. 3. The effect of inhibitors of DG metabolism on AII-induced 12-HETE levels. Values are mean * SE from four experiments performed in triplicate. * p < 0.05 vs. basal; * * p < 0.02 vs. AH; * * * p < 0.02 vs. AH.
ng, p < 0.02 vs. AII). Similar to its effects on aldosterone, RHC 80267 blocked the potentiating effect of R59022 bringing 1ZHETE levels back to control (AI1 + RHC 80267 + R59022,7.5 + 0.8 ng, p < 0.01 vs. AI1 + R59022). The effect of 12-HETE addition on AII-induced aldosterone during DG Iipase inhibition Table 1 depicts the effect of 12-HETE addition on AII-induced aldosterone synthesis during DG lipase inhibition. AII-induced action during inhibition by RHC 80267 is completely restored by the addition of the 12-LO product 12-HETE (Table 1). The effect of AII on DG levels Fig. 4 depicts the time course of AII-induced DG levels in glomerulosa cells labeled with [3H]AA. We observed that AI1 (10e9 M) stimu-
The effect of AII and the inhibitors of DG metabolism on DG levels Fig. 5 shows the effect of AI1 alone and with inhibitors of DG metabolism on [3H]AA-labeled DG levels. Results shown are from 10 min experi-
* ** l/y -
I
H
Control
Au10-9M
I
J
A
TlME(MIN.)
Fig. 4. Time course of AII-stimulated diacylglycerol formation. The results are percent control radioactivity in DG fraction and are expressed as meanf SE from 3-5 experiments performed in duplicate or triplicate. * p < 0.04 vs. control; **p < 0.01 vs. control. Incubation and lipid extractions were carried out as described in Materials and Methods.
78
5
1707
c Y z
160-
x z
150-
f _ 5 F
140
Y
d J 0 E 6
120
110
0 8 2
100
Lz 130 : ! AIIIlO-WI
All+ RHC80267
All + I359022
All + R59022+ RHC80267
Fig. 5. The effect of AI1 alone and with inhibitors of DG metabolism on [3H]DG levels. Results shown are data obtained from 10 min incubations. Values are mean k SE from 3-5 experiments performed in duplicate or triplicate. *p < 0.01. RHC 80267 and R59022 were used at lo-’ M and 10m5 M resp.
ments. All stimulated DG levels to 137 k 4% of basal ( p < 0.001). RHC 80267 used alone did not significantly alter the effect of All on DG levels (All + RHC 80267,131 + 3%, p < 0.001 vs. basal). However, R59022 had a slight potentiating effect vs. All alone (All + R59022, 148 + 5% basal). In contrast as seen in Fig. 5 the combination of the two inhibitors significantly potentiated the All effect on stimulation of DG (167 + 6% basal, p < 0.01 vs. All). Discussion
DG plays an important role in the signal transduction of All action in the adrenal glomerulosa cell. DG can directly activate protein kinase C. However, DG can also be converted to phosphatidic acid by DG kinase or lead to AA release via DG lipase. In a preliminary study, a DG lipase inhibitor reduced All-induced aldosterone synthesis in human glomerulosa cells suggesting that AA release from DG may also be important in All action (Natarajan et al., 1988b).
In the present report we found that All stimulated DG levels in a biphasic fashion with peaks at 30 s and 10 min. These are the first results of DG levels in rat glomerulosa cells and are similar to those reported earlier in bovine cells (Kojima et al., 1984; Hunyady et al., 1990). The mechanism and role of this biphasic DG response after All stimulation are not clear. However, previous studies suggest that this may be due to a shift in substrate requirement from phosphatidylinositol biphosphate to phosphatidylinositol (Griendling et al., 1986). Sutherland and Amin (1982) first described RHC 80267 as a potent and selective inhibitor of canine platelet DG-lipase. In more recent studies RHC 80267 has been used to evaluate the role of AA release in DG action in endocrine tissues. Several reports have shown that RHC 80267 can inhibit prolactin release (Canonico et al., 1985; Camoratto and Grandison, 1987; Judd et al., 1988). Chang et al. (1988) showed that DG metabolism via DG lipase is an important step in gonadotropin-releasing hormone-induced stimulation of luteinizing and follicle-stimulating hormone secretion. In the latter study, RHC 80267 action was specific since it did not alter phospholipase A, or C, protein kinase C or Ca*+ responses in cells. In our earlier studies with human adrenal cells, RHC 80267 action at 5 PM was specific to All and Kf-induced aldosterone synthesis and did not alter ACTH effects (Natarajan et al., 1988b). In the current study, RHC 80267 synthesis was effective at concentrations as low as lo-’ M in blocking All-induced aldosterone and 12-HETE. We have also shown that this dose of RHC 80267 inhibits AII-induced release of [‘H]AA from prelabeled rat adrenal glomerulosa cells (unpublished observations). RHC 80267 at lo-’ M concentration also inhibited K+-induced aldosterone synthesis. These results are similar to our previous studies in human adrenal cells (Natarajan et al., 1988b). Preliminary studies also suggest that K+ induces [3H]AA release and that K+-induced aldosterone synthesis is markedly potentiated in rat adrenal cells incubated in the presence of 1 PM arachidonic acid (unpublished observations). Our earlier studies have shown that K+ does not induce 12-HETE formation (Nadler et al., 1987). Therefore, AA
79
itself or other non-LO metabolites of AA may play an important role in Kf-mediated aldosterone synthesis. Additional studies will be required to evaluate these effects. In the current study we did not evaluate the effect of RHC 80267 on ACTH action. We previously showed that in human adrenal cells RHC 80267 did not alter ACTH-induced effects (Natarajan et al., 1988b). However, very recent evidence (Cozza et al., 1990a, b) suggests that ACTH can induce the de novo synthesis of DG in bovine adrenal cells, suggesting that there may be species variation in ACTH action. Therefore, additional studies will be required to evaluate whether in the rat, ACTH action involves DG formation and AA release. R59022 has been reported to be a selective inhibitor of DG kinase in both intact human platelets and human red cell membranes (DeChaffoy de Courcelles et al., 1985). In thrombinactivated platelets, addition of R59022 resulted in a marked elevation of DG levels, decreased formation of phosphatidic acid and increased protein kinase C activity. The IC,, for DG kinase inhibition was 3.8 X lop6 M and approximately 80% inhibition was obtained at 10e5 M. Most studies with R59022 have employed 10m5 M concentration. R59022 has been used by several workers as a valuable tool for the amplification of DG signals in studies related to mechanisms of signal transduction. Muid et al. (1987) employed R59022 to determine the important role of protein kinase C and arachidonate in the signal transduction of the respiratory burst in the neutrophil. It has also been useful in elucidating the contribution of the protein kinase C pathway in long-term mitogenesis (Morris et al., 1988). In the present study, the DG kinase inhibitor, R59022, caused a marked potentiation of the AI1 effects on aldosterone and 12-HETE. RHC 80267 and R59022 are both inhibitors of DG metabolism, a process that should lead to build up of DG which in turn should lead to increased protein kinase C activity and AI1 action. However, only R59022 potentiated AI1 action whereas RHC 80267 was inhibitory. We hypothesized that RHC 80267 was blocking AI1 action due to its inhibitory effect on AA and 12-HETE formation. In order to test this, we examined the effect of these
inhibitors on AII-induced 12-HETE formation. Similar to its effect on aldosterone, RHC 80267 also blocked AII-induced 12-HETE levels, while R59022 caused a marked potentiation of the 12-LO product. Furthermore, RHC 80267 also prevented the potentiating effect of R59022 on 12-HETE formation. 12-HETE addition could restore the effects of AI1 during DG lipase inhibition, thus supporting the specificity of action of RHC 80267. These results are consistent with the hypothesis that 12-HETE formation from DG is an important step in AI1 action in the adrenal. Therefore, increasing DG levels potentiates AII-mediated aldosterone synthesis only when DG can form AA products via DG lipase. We measured [‘H]DG levels in the presence of AI1 and these inhibitors in order to provide additional support for the action of these agents. Neither the inhibitors RHC 80267 nor R59022 significantly altered AII-induced stimulation of DG at 10 min. Moreover, the combination of the two inhibitors significantly potentiated AI1 effect on DG even though this same combination was inhibitory on AII-induced aldosterone and 12HETE. Thus the inhibitory effect of RHC 80267 on AII-induced aldosterone and 12-HETE levels was not due to a blockade of DG formation, but instead due to a blockade of AA release. The major sources of AA in cells are from phosphatidylcholine, phosphatidylethanolamine or phosphatidic acid by the action of phospholipase A, (Bills et al., 1977; Lapetina et al., 1981) or from phosphoinositides by the sequential action of phospholipase C and DG lipase (Bell et al., 1979; Chau and Tai, 1981). Previous studies have shown that in the adrenal, AI1 does not activate phospholipase A, (Hunyady et al., 1982). Thus, DG via DG lipase could provide an important source of AA and 12-HETE in adrenal glomerulosa cells. Our earlier studies have shown the importance of 1ZHETE in AII-mediated aldosterone synthesis in rat and human adrenal glomerulosa cells (Nadler et al., 1987; Natarajan et al., 1988a). The current study confirms that the origin of 12-HETE is from AA derived from DG. We have proposed that AA metabolites of the 12-LO pathway could facilitate the action of protein kinase C in mediating AI1 action. The possible mechanisms by which 12-HETE could achieve
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this is by the activation of protein kinase C directly or by the activation of Ca2+ or both. Several fatty acids including HETEs have been shown to activate protein kinase C (Murakami and Routtenberg, 1985; Hansson et al., 1986) and recently AA and 12-HETE have been shown to activate the r-subspecies of protein kinase C directly in a Ca’+-independent manner (Shearman et al., 1989). Seifert et al. (1988) have suggested that fatty acids and DG may be synergistically involved in hormonal stimulation of protein kinase C. We have also shown using fura- Ca*+ measurements in rat cells that 12-HETE also stimulates Ca*+ in a manner similar to AI1 (Stern et al., 1988). Further studies will be needed to fully evaluate the role of protein kinase C and Ca2+ release by 12-HETE in mediating AI1 action in the adrenal. In summary, this study suggests that DG generated by receptor activation plays a central role in AI1 action, not only as an endogenous activator of protein kinase C, but also as a precursor of active lipoxygenase metabolites of AA that mediate AI1 action. Acknowledgement The authors wish to thank Ms. Jenny Yang for her secretarial assistance in typing the manuscript. References Abe, K. and Kogure, K. (1986) J. Neurochem. 47, 577-582. Bell, R.L., Kennerly, D.A., Stanford, N. and Majerus, P.W. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3238-3241. Berridge, M.J. (1984) B&hem. J. 220, 345-360. Berridge, M.J. and Irvine, R.F. (1984) Nature 312, 315-321. Bills, T.K., Smith, J.B. and Silver, M.J. (1977) J. Clin. Invest. 60, 1-6. B&h, E.G. and Dyer, W.J. (1959) Can. J. B&hem. Physiol. 37, 911-917. Braley, L.M. and Williams, G.H. (1977) Am. J. Physiol. 233, 402-406. Camoratto, A.M. and Grandison, L. (1987) Life Sci. 40, 275281. Canonico, P.L., Cronin, M.J. and Macleod, R.M. (1985) Life Sci. 36, 997-1002. Chang, J.P., Morgan, R.O. and Catt, K.J. (1988) J. Biol. Chem. 263. 18614-18620.
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