in Rat Luteal Cells, and Effects on Steroidogenesis - American Journal ...

Articles in PresS. Am J Physiol Endocrinol Metab (February 14, 2006). doi:10.1152/ajpendo.00205.2005

Localized Accumulation of Angiotensin II and Production of Angiotensin-(1-7) in Rat Luteal Cells, and Effects on Steroidogenesis.

John R. Pepperell1,2, Gabor Nemeth1, Yuji Yamada1, Frederick Naftolin1 and Maricruz Merino3

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Department of Obstetrics and Gynecology, Yale University Medical School, 333 Cedar St, New Haven, CT 06520.

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Department of Pathology, 3Department of Pediatrics, Brown University Medical School, Women & Infants Hospital, 101 Dudley St, Providence RI 02905.

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Running Title: Luteal Angiotensins and Steroidogenesis

Author for correspondence: J.R. Pepperell, Department of Pathology, Brown Medical School, Women & Infants Hospital, Providence, RI 02905. E-Mail: [email protected]

Copyright © 2006 by the American Physiological Society.

Luteal Angiotensins and Steroidogenesis

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ABSTRACT These studies aim to investigate subcellular distribution of angiotensin II (Ang II) in rat luteal cells, identify other bioactive angiotensin peptides, and investigate a role for angiotensin peptides in luteal steroidogenesis. Confocal microscopy showed Ang II distributed within the cytoplasm, and nuclei of luteal cells. HPLC analysis showed peaks that eluted with the same retention times as Ang(1-7), Ang II and Ang III. Their relative concentrations were: Ang II

Ang-(1-7) >>Ang III, and

accumulation was modulated by quinapril, an inhibitor of the angiotensin converting enzyme (ACE), Z-proprolinal (ZPP) an inhibitor of prolyl endopeptidase (PEP), and parachloromercurylsulfonic acid (PCMS) a sulfhydryl protease inhibitor. Phenylmethylsulfonyl fluoride (PMSF) a serine protease inhibitor did not affect peptide accumulation. Quinapril, ZPP, PCMS, PMSF, losartan, the angiotensin receptor type 1 (AT1), and PD123319, the AT2, receptor antagonists were used in progesterone production studies. ZPP significantly reduced LH-dependent progesterone production (p < 0.05). Quinapril plus ZPP had a greater inhibitory effect on LH-stimulated progesterone than either inhibitor alone, but this was not reversed by exogenous Ang II or Ang-(1-7). Both PCMS and PMSF acutely blocked LH-stimulated progesterone, and PCMS blocked LH-sensitive cyclic AMP accumulation. Losartan inhibited progesterone production in permeabilized, but not intact luteal cells that was reversed by Ang II. PD123319 had no significant effect on luteal progesterone production in either intact or permeabilized cells. These data suggest that steroidogenesis may be modulated by angiotensin peptides that act in part through intracellular AT1 receptors.


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INTRODUCTION The formation of Ang II occurs as the result of a cascade reaction that is initiated by activation of prorenin to renin. The substrate angiotensinogen is hydrolyzed by renin, releasing the decapeptide, Ang I that in turn is substrate for angiotensin converting enzyme (ACE). ACE hydrolysis of Ang I results in the formation of the octapeptide Ang II, considered the major biologically active peptide of the cascade, and its proteolysis produces Ang III (Ang-(2-8)). Historically, Ang II was considered an adrenal hormone restricted to renal function, but evidence that Ang II is produced by other tissues, including the ovaries, began to emerge in the 1980’s. The demonstration of ovarian angiotensinogen, Ang II receptors, and renin and ACE activity has been reviewed (52, 82), and this ovarian renin angiotensin system (OVRAS) appears to be under gonadotrophin control. Prorenin/renin and Ang II/Ang III immunoreactivity is present in follicular fluid of women at significantly higher concentrations than in serum, indicating local ovarian synthesis (21, 39), and these concentrations increase following gonadotrophin treatment, or during the mid-cycle gonadotrophin surge (39, 40, 63). Similarly in the rat, gonadotrophin regulation of the OVRAS is implicated since granulosa cells of immature developing follicles show no immunostaining for renin or Ang II, but at the time of the gonadotrophin surge, granulosa cells of the preovulatory follicle stain intensely for both these antigens (38). However the role of the OVRAS in ovarian function remains to be clarified. For example, Ang II mediates ovulation in the rat (50, 53), but Ang II receptors in preovulatory follicles of this species have not been demonstrated (65). Angiotensin II receptors are classified as type 1 (AT1) or type 2 (AT2) based on their pharmacology (11, 77), and in the rat, the AT2 receptor is associated with atretic follicles (34), suggesting a role in follicular atresia. On the other hand, in the rabbit, AT1 is present in granulosa cells and thecal cells of preovulatory follicles (18), and in this species Ang II mediates meiotic maturation and stimulation of ovulation in part through prostaglandin


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production (35, 83). A role for Ang II in ovarian steroidogenesis also requires clarification. In vivo studies using a microdialysis system showed stimulatory effects of Ang II on progesterone output in the newly formed bovine corpus luteum (32), but at late stages of luteal formation (day 15), Ang II has luteolytic effects (72). In the case of bovine luteal cells in culture, Ang II inhibits cholesterol side-chain cleavage activity and reduces progesterone production (66). Inhibitory effects of Ang II on LH-stimulated steroidogenesis also occur in cultured pig and human granulosa cells (28, 37). In cultured rat luteal cells, we suggested that Ang II and the natural luteolysin PGF2 could work together to reduce progesterone (79), and more recently, a positive feedback loop between Ang II, endothelin, and PGF2 , produced within the luteal environment, has been proposed to be key in functional and structural luteolysis of the bovine corpus luteum (72). Until recently, the renin-angiotensin system was considered to produce Ang II as the only biologically active molecule of the cascade. This view is challenged by the demonstration that additional biologically active angiotensin fragments are produced from angiotensinogen (reviewed in (13, 20)). ACE hydrolysis of Ang I results in the formation of Ang II, that is further proteolytically degraded by aminopeptidase A to Ang III (Ang-(2-8)), or by aminopeptidase D to Ang IV (Ang-(3-8)), and other fragments (57). An additional pathway for lysis of Ang I is via prolyl endopeptidase (EC 3.4.21.26) (PEP), or neutral endopeptidase (EC 3.4.24.11) (NEP) resulting in the formation of Ang-(17). However, Ang-(1-7) can also be directly formed from Ang II by PEP or prolyl carboxypeptidase activity. Thus, in addition to Ang II, other bioactive angiotensin peptides, including Ang III, Ang-(17), and Ang IV, can be produced by the renin-angiotensin cascade (20, 27). Further biological complexity of the renin-angiotensin system is bestowed by specific angiotensin peptide receptors. The type 1 (AT1), and the type 2 (AT2) Ang II receptor, are recognized to mediate different actions (11,


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78), while receptors for Ang IV and Ang-(1-7) are also implicated since they often have opposing effects to those of Ang II (11, 16, 20, 43), and recently the orphan receptor, Mas was proposed to be the Ang-(1-7) receptor (62). Clearly, a combination of angiotensin peptides and their receptors in any given tissue can confer a highly complex regulatory system, and such complexity is indicated in the ovary (13). It is the hypothesis of this study that since the corpus luteum is a source of Ang II (32, 48), other angiotensin peptides are also produced, and that different angiotensin peptides may differentially regulate luteal steroidogenesis. Therefore, we intend to determine whether Ang-(1-7) is formed by the corpus luteum, and investigate the effects of angiotensin peptides on progesterone production. METHODS Animals Immature (26-27 days old) female rats (CD strain, Charles River Laboratories, Wilmington, MA) were injected s.c. with 50 IU PMSG (Gestyl, Organon Pharmaceuticals, West Orange, NJ) to induce superovulation. Fifty four hours later, 25 IU human CG (hCG) (Wyeth-Ayerst Laboratories, Rouses Point, NY) was injected to induce ovulation and pseudopregnancy. All procedures were in accordance with institutional animal care and use committee guidelines. Luteal cell isolation Rat luteal cells were isolated by methods previously described (51). Briefly, 5 days after injection of hCG, the ovaries were removed and finely minced with a razor blade. The ovarian fragments were suspended in 5 ml Ca2+-free Minimal Essential Medium (medium 1, 1380, GIBCO, Grand Island, NY) containing 0.1% bovine serum albumin (BSA, GIBCO), 2000 IU collagenase (Worthington Biochemical Corp., Freehold, NJ) and 3000 IU deoxyribonuclease (DNAase, Worthington) per g tissue, for 1 h at 37o C under 95% air-5% CO2. The isolated luteal cells were enriched by density gradient sedimentation on a discontinuous density gradient (51) (Percoll,


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Pharmacia Fine Chemicals, Uppsala, Sweden). The cells were washed with 10 ml medium 1, collected by centrifugation and resuspended in Dulbecco's Modified Eagle's/Ham's F12 Medium at 0.5-1.0 million cells/ml containing 0.1% FCS, penicillin (100 U/ml), streptomycin (0.1 µg/ml), and amphotericin-B (0.25 µg/ml; all Gibco). Cell number was determined by means of hemocytometer, and cell viability was greater than 90 %, as assessed by exclusion of trypan blue dye. Immunohistochemistry Luteal cell suspensions in DMEM containing BSA (0.1%) were cultured in Lab-Tek culture slides for 24 hours, after which they were fixed with 4% paraformaldehyde in DPBS pH 7.4 for 1 hour. Inhibition of endogenous luteal peroxidase was achieved with 5% hydrogen peroxide (1 hour). The cells were washed with DPBS, incubated with 0.1% triton-x in DPBS for 30 minutes at room temp, and then washed twice (5 min each) with DPBS. To recover antigenicity, cells were placed in a microwave oven for 1min at maximum power in citrate buffer (50 mM, pH6.0) (54), and then(54) washed once with DPBS for 5 minutes. Non-specific binding sites were blocked with Power BlockTM (BioGenex, San Ramon, Ca) incubated at 37o C for 20 minutes, followed by washing twice with DPBS for 5 min. The cells were incubated overnight at 4o C with Ang II antibody (diluted 1:300 in DPBS containing BSA (3 %)) in a humidified, chamber. Before secondary labeling with streptavidin/DAB, the cells were washed in DPBS (1 x 5 min; 1 x 30 min, 1 x 10 min). The cells were then incubated with Multilink (BioGenex) for 10 min, washed three times (5 min each) in DPBS, followed by incubation with Label (BioGenex) for 15 min. Subsequently, cells were incubated with Substrate (BioGenex) for 5 minutes. The cells were counterstained with hematoxylin for 1 min, and NH4OH for 3 min, followed by a wash with deionized H2O before mounting. In the case of confocal and fluorescence microscopy studies, paraformaldehyde fixed cells were incubated with triton-x 100 (0.1%) in DPBS for 30 min, and washed three times with DPBS (10


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minutes each wash). Cells were incubated for 1h at 37o C with DPBS containing 5% normal rabbit serum and 3% BSA (blocking solution), followed by three washes with DPBS (each 10 minutes). Immunolabeling was achieved with rabbit anti-Ang-II antibody (1:400) in DPBS containing BSA (3%) incubated overnight at 4o C, followed by 3 washes with DPBS (15 minutes each wash) and incubation for 45 minutes at room temp with fluorescein-labeled goat anti-rabbit IgG FAB (Cappel, Irvine, CA) (diluted 1:100 in blocking solution). Unbound label was removed by washing the cells three times with blocking solution, followed by two washes with DPBS alone. The slides were mounted with Slowfade (Molecular Probes, Portland, OR) containing propridium iodide. HPLC Analysis Angiotensin peptides were resolved by HPLC, based on published methods (9). Isolated luteal cells were aliquotted to tubes, centrifuged (1200 x g, 10 minutes) and resuspended with intracellular (IC) buffer (KCl 110 mM; NaCl 10 mM; KH2PO4 1 mM; KHCO3 5 mM; HEPES 20 mM, pH 7.1) containing triton-x (0.1%) at 4o C. The cell suspension was sonicated on ice for 10 seconds at 50 % cycle with a miniprobe. Aliquots were added to tubes containing in a final volume of 100 µl, quinapril, ZPP, PCMS or PMSF (each 100 µM) and angiotensinogen (25 µM). Following incubation for 30 minutes the cells were extracted for HPLC. Luteal cells were suspended in 80% ethanol containing 0.1N HCl and incubated at -20o C overnight, followed by centrifugation (20k x g) for 20 minutes. The supernatant was diluted 1:1 with 1% heptafluorobutyric acid (HFBA) and kept at 4o C for 6 h before centrifugation (20 min, 20K x g). The supernatant was removed, diluted 1:1 with 0.2% HFBA, and applied to an activated Sep-Pak C18 cartridge (Waters Associates, Milford, MA). After washing with 0.2% HFBA, the columns were eluted with 10 ml 80% methanol, 0.2% HFBA into a tube containing 100 µl 10% chick egg albumin. The eluate was dried in a speed vac (Savant), resuspended with acetonitrile (80%)/HFBA (0.13%) and centrifuged at 1300 x g for 20 min. Samples


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were filtered (0.22 µm) before injection into the sample port of Waters Delta Prep 3000 HPLC system (Waters Associates, Milford, MA), equipped with a Nova-Pak C18 reverse phase column (3.9 x 150 mm). Elution solvents were: mobile phase A, 0.13 % HFBA; and mobile phase B, 80% acteonitrile/0.13% HFBA. The column was equilibrated with 10 column volumes of 75% mobile phase A and 25% mobile phase B at a flow rate of 1 ml/min. Samples were applied to the column dissolved in 20% mobile phase B, and angiotensin peptides eluted with a 30 minute gradient (curve 4 of the Waters automatic gradient controller) to 50% mobile phase B. The eluted peptide peaks were detected by an UV-detector (220 nm) and plotted to a recorder. The area under the peaks of fractions that migrated with the same retention times as synthetic standards for Ang-(1-7), Ang II and Ang III were resolved to estimate the amounts of peptide. Extraction efficiency was calculated from extraction of known concentration of peptides, and comparison of the elution peaks with non-extracted standards. Cell Incubations Isolated luteal cells were washed with 10 ml medium 1, collected by centrifugation and resuspended in Dulbecco's Modified Eagle's/Ham's F12 Medium at 0.5-1.0 million cells/ml containing 0.1% BSA, penicillin (100 U/ml), streptomycin (0.1 µg/ml), and amphotericin-B (0.25 µg/ml; all Gibco) (DMEM). Whole Cells: Luteal cells were incubated in 12 x 75 cm glass assay tubes at a cell concentration of 1-2 x 105 cells/ml DMEM. Experiments adopted a paradigm of a pre-incubation with protease inhibitors for 30-60 minutes as specified in legends, followed by an acute 1 h incubation with and without LH (100 ng/ml). Cell incubations for ACE assays were carried out in 24 well plates at an initial cell concentration of 1 x 106 cells/ml. Permeabilized Cells : Permeabilization of rat luteal cells was based on published methods that describe the use of saponin to allow exogenous bioactive molecules access to the intracellular milieu


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with continued cell function (36, 76). Luteal cells were washed with intracellular (IC) buffer (containing in mmoles: HEPES, 20; KCl, 110; NaCl, 10; KH2PO4, 1; KHCO3, 5; MgCl2, 0.3; pH 7.1), centrifuged and resuspended at 1 x 107 cells/ml with IC buffer supplemented with MgCl2 (3.3 mM), ATP (4.4 mM) and saponin (0.1 mg/ml). The cells were incubated for 2-5 minutes at 20o C, when permeabilization was achieved with 100 % cells taking up trypan blue. The permeabilized cells were centrifuged and resuspended with IC buffer containing MgCl2 (3.3 mM) and ATP (4.4 mM), creatine phosphokinase (10 U/ml) and creatine phosphate (20 mM), and aliquotted at 2 x 10 5 cells/ml to tubes in the presence of LH (100 ng/ml), and in the absence and presence of Ang-(1-7), Ang II, losartan, or PD123319. Following incubation for 1 h at 37o C, the tubes were immersed in an 80o C water bath for 10 minutes before storage at –20o C awaiting progesterone RIA. Progesterone and cyclic AMP Radioimmunoassay Total progesterone and cyclic AMP were determined in combined cells and media by specific radioimmunoassay, as previously described (51). Angiotensin Converting Enzyme Activity Angiotensin converting enzyme (ACE) activity was measured by means of o-aminobenzoylGly-p-Nitro-Phe-Pro (ABGP) which forms a fluorescent product upon cleavage by ACE (7). Luteal cells were incubated with Dulbecco's phosphate buffered saline (DPBS, 500 µl) containing glucose (1 g/l) and ABGP (1 mM) dissolved in dimethyl sulfoxide (DMSO, final concentration 0.5%). Cells were separated from the media at the end of the incubation period by centrifugation (5 minutes 800 x g). Aliquots (200 µl) of supernatant were added to 2 ml of Tris (0.2 M, pH 8.2) containing ethylene diaminetetraacetic acid (EDTA, 0.1 M) at 4o C, and the fluorescence in the media was measured with a fluorimeter (Deltascan PTI, Photon Technology International, Brunswick, NJ) (excitation 360 nm,


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emission 460 nm). Fluorescence of substrate blanks that contained all reagents except for cells were subtracted from experimental reactions. Preliminary experiments with luteal cells and ABGP (1 mM) determined a linear time-dependent increase in fluorescence of product over 12 h, and that substrate concentration was not limiting for product formation in the assay. Statistical Analysis Statistical difference between multiple treatments was determined by one way analysis of variance (ANOVA) or two-way ANOVA and specific differences among groups identified by post-hoc tests, Student-Newman-Keuls or paired t-test. A significant difference was established at the level of p < 0.05. Each experiment included at least three replications per treatment, and data are presented as the mean + standard error of the mean (SEM) of “n” experiments as expressed in the Figure legends. Hormones, Drugs and Reagents Ovine luteinizing hormone (oLH#26) was supplied by NIDDK. ZPP was generously donated by Dr. K.B. Brosnihan (Wake Forest, NC). Quinapril and PD123319 were obtained from Parke-Davis (Ann Arbor, MI), and Losartan was obtained from Merk, Sharpe and Dohme (Rahway, NJ). Angiotensin II, porcine renin substrate tetradecapeptide (angiotensinogen) and all other chemicals were purchased from Sigma Chemical Co. (St. Louis MO).

RESULTS Figure 1 shows images of luteal cells obtained under differential interference contrast (DIC) microscopy. The presence of Ang II-immunoreactivity was confirmed in luteal cells by the presence of intense dark immunostaining, but when the primary antibody was omitted in negative control incubations, no immunostaining was apparent. The distribution of immunolabeled Ang II in cultured luteal cells was further studied with confocal fluorescence microscopy, and Figure 2 is a composite


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image formed from six stacked images obtained at 1 µm sections. Ang II is not only distributed throughout the cytoplasm of the luteal cells, but also intense patches of Ang II are present within the nuclei. (No labeling of Ang II in rat luteal cells was observed in the absence of anti Ang II primary antibody). To further characterize the presence of angiotensin peptides in luteal cells we used HPLC analysis to identify and estimate the relative amounts of the bioactive angiotensin peptides Ang-(1-7), and Ang II and Ang III. Figure 3 (A, upper panel) shows an HPLC elution profile of synthetic angiotensin standards that were extracted from IC buffer, and the elution of Ang-(1-7), Ang II and Ang III occurred at 16.4 ± 0.2 minutes, 25.1 ± 0.2 minutes and 26.1 ± 0.2 minutes, respectively. Integration of the area under the peaks of absorbance of known concentrations of peptide standards and linear regression analysis found a highly significant correlation (p < 0.0001) between peptide concentration and integrated area (r2 = 0.960, 0.989, 0.977, for Ang-(1-7), Ang II and Ang III, respectively). The efficiency of extraction of peptides was calculated by comparing the integrated areas obtained from extracted standards with those areas obtained from non-extracted standards. Recovery was 92.9 ± 5.0 %, 85.5 ± 8.8 % and 83.3 ± 5.3 % (n = 4) for Ang-(1-7), Ang II and Ang III, respectively. Extracts from luteal cells displayed peaks that correlated with Ang-(1-7), Ang II and Ang III, but since we wished to investigate angiotensin processing pathways and effects of protease inhibitors, we incubated luteal cells with angiotensinogen as described in the Methods Section, to prevent substrate depletion effects that would compromise interpretation of the data. Figure 3 (B, lower panel) shows a luteal cell chromatogram with absorbance peaks that correlated with Ang-(1-7), Ang II and Ang III, and the relative amounts of these peptides were determined to be: Ang-(1-7), 586.5 ± 63.1 pmoles/mg; Ang II, 622.1 ± 87.2 pmoles/mg; and Ang III, 244.1 ± 55.9 pmoles/mg (n=8). Protease inhibitors that may modulate angiotensin peptide include quinapril, a specific non-


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peptide, non-sulfhydryl, competitive inhibitor of ACE (12); PCMS, a sulfhydryl protease inhibitor; ZPP, an inhibitor of PEP (61), and PMSF, a serine protease inhibitor. Luteal cells were incubated for 60 minutes in the presence of each inhibitor (100 µM) in DMEM containing BSA (0.1%), and then centrifuged and resuspended with IC buffer before a secondary incubation with protease inhibitor and angiotensinogen as described in the Methodology Section. Peptides were extracted, and subjected to HPLC. Table 1 shows the effects of protease inhibitors on accumulation of angiotensin peptides by luteal cells. Quinapril significantly reduced Ang III levels (p < 0.05), but a trend of reduced Ang II did not reach statistical significance. Ang-(1-7) was also unaffected by quinapril. ZPP significantly increased accumulation of Ang II (p < 0.05), but was without statistically significant effect on Ang-(17) or Ang II levels. When cells were incubated with ZPP plus quinapril combined, the accumulation of Ang-(1-7) was significantly reduced (p < 0.05), but Ang II and Ang III were not significantly different from control levels. The sulfhydryl protease inhibitor PCMS significantly reduced accumulation of Ang-(1-7), Ang II and Ang III, but no effect of PMSF on peptide accumulation was detected. Figure 4 summarizes data of studies that investigated the effects of protease inhibitors on basal and LH-stimulated steroidogenesis. Under basal conditions, quinapril and PMSF had no statistically significant effects; however PCMS at higher concentrations (100 µM) significantly reduced progesterone production (p < 0.05). ZPP on the other hand, enhanced progesterone output when compared to the effects of the other protease inhibitors, and at 100 µM the level of progesterone production was elevated compared to that of its control group (p< 0.05). In the case of LH-stimulated cells, the protease inhibitors tended to reduce progesterone production. PCMS and PMSF induced a dose-dependent inhibition that reached approximately 70 % and 50 % inhibition, respectively at the maximally tested concentration (100 µM). The effects of quinapril and ZPP were more subtle than those of PCMS and PMSF: ZPP (100 µM) inhibited LH-driven progesterone production approximately


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15 % compared to the control group (p < 0.05); however, progesterone production in the presence of quinapril (100 µM) was not statistically different from control levels, although an approximate 10 % reduction in its output was detected when compared to cells incubated with 10 µM quinapril (p < 0.05). Other protease inhibitors were tested at concentrations up to 100 µM and found to have no effect on basal or LH-stimulated progesterone production including benzamidine, pepstatin and aprotinin (data not shown). The effects of the protease inhibitors on LH action may be mediated through the second messenger cyclic AMP (cAMP), therefore we investigated their effects on cAMP accumulation, and the results from these experiments are summarized in Figure 5. Under basal conditions, no statistically significant dose-response effects were detected within the protease inhibitor treatments. However among the treatment groups, cells treated with PCMS accumulated less cyclic AMP than did cells treated with quinapril, ZPP, or PMSF (pAng III.


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These observations suggest that Ang II and Ang-(1-7) are significant products of angiotensinogen processing in the rat corpus luteum. The formation and regulation of Ang II and Ang-(1-7) in the ovary may have consequences for health and disease. Ang II and Ang-(1-7) are frequently counterregulatory: Ang II is a potent vasoconstrictor, stimulates mitogenesis, and has angiogenic effects, while Ang-(1-7) is a vasodilator, reduces mitogenic activity, and opposes angiogenesis (5, 19, 33, 41, 69). Thus, the balance of production of these two peptides may play a role in formation of the corpus luteum, and also play a role in ovarian pathologies such as ovarian cancer, thereby underlining the importance to understand their biochemical pathways. PCMS, the sulfhydryl protease inhibitor significantly inhibited accumulation of Ang-(1-7), Ang II and Ang III. The broad inhibitory effects of PCMS on angiotensins are likely due to the sulfhydryl groups of ACE that are susceptible to PCMS (14, 23), thereby inhibiting hydrolysis of Ang I to Ang II, that can be precursor for both Ang-(1-7) and Ang III. In addition, a sulfhydryl containing peptidase identified in a neuroblastoma-glioma cell line (8) may also occur in luteal cells, and contribute to PCMS-sensitive inhibited Ang-(1-7) accumulation. The serine protease inhibitor PMSF was without effect on accumulation of angiotensin peptides. In some tissues PMSF inhibits activation of prorenin to renin, and also reduces Ang II accumulation (60, 70), but the present data does not identify a role for serine protease activity in angiotensinogen processing in luteal cells. The broad specificity of PCMS and PMSF directed studies with quinapril and ZPP that represent more specific inhibitors of proteases of angiotensin processing. Quinapril specifically inhibits ACE mediated hydrolysis of Ang I to Ang II (12), and ZPP inhibits the PEP pathway that hydrolyses Ang I or Ang II to Ang-(1-7) (8, 61). Treatment of luteal cells with quinapril significantly reduced accumulation of Ang III, but a trend of reduced accumulation of Ang II did not reach statistical significance. These findings suggest that the proteolytic cascade of Ang I to Ang II to Ang III via ACE activity is not a major pathway in luteal


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cells. Alternative angiotensinogen processing that can give rise to Ang II production includes direct proteolysis of angiotensinogen to Ang II, independent of renin and ACE activity (20); in addition chymase/cathespsin G appears the major enzyme that converts Ang I to Ang II in heart tissue (29). It is not clear whether a similar pathway may occur in luteal cells since chymase/cathespsin G is a serine protease, and in our studies we found no significant effect of the serine protease inhibitor PMSF on accumulation of Ang II. Further studies are necessary to identify whether pathways, in addition to ACE, generate Ang II from Ang I in the rat corpus luteum. However, alternative ovarian pathways of Ang II formation could explain the lack of effect of competitive ACE inhibitors on ovulation observed in studies with rats and rabbits (15, 59), species known to be dependent on Ang II for ovulation (50, 84). Under basal conditions, incubation of luteal cells with ZPP that inhibits PEP statistically enhanced Ang II accumulation, but did not affect accumulation of Ang-(1-7) or Ang III. PEP can utilize Ang II as substrate to form Ang-(1-7) (61, 81), thus ZPP-enhanced accumulation of Ang II may be explained by reduced metabolism of Ang II to Ang-(1-7). Therefore, enhanced levels of Ang II in ZPP-treated luteal cells suggest the presence of a hydrolytic pathway that utilizes PEP hydrolysis of Ang II to produce Ang-(1-7) in this tissue. Furthermore, inclusion of quinapril with ZPP reduced Ang(1-7) and abrogated the increased accumulation of Ang II, indicating ACE hydrolysis of Ang I to Ang II and its PEP-mediated hydrolysis to Ang-(1-7) may occur in luteal cells. Although the net accumulation of Ang-(1-7) was unaffected by ZPP by itself, we cannot rule out that reduced levels of production coupled with a low turnover maintains Ang-(1-7) levels in luteal cells. In addition, Ang-(17) production directly from Ang I via NEP that is independent of Ang II and ACE activity can occur in some cell types (61), and a similar route in luteal tissues may contribute to accumulated Ang-(1-7) levels. The effect of alternative pathways of angiotensinogen processing includes the result that


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shutting down one pathway can drive enhanced accumulation of other angiotensin peptides. It is conceivable that in vivo, different pathways may operate in specific conditions of health or disease. Indeed, levels of circulating Ang-(1-7) and Ang II increase during pregnancy in the human, and a change in the ratio of their concentration is implicated in the onset of pre-eclampsia (45), but it remains to be determined whether the imbalance in their ratios arises from differential angiotensin peptide production by the corpus luteum of pregnancy. The effects of the protease inhibitors on basal steroidogenesis correlated with their effects on Ang II accumulation. Quinapril and PMSF had no significant effects on progesterone production or Ang II accumulation; PCMS at higher concentrations inhibited progesterone and Ang II levels; while ZPP enhanced basal progesterone production and Ang II accumulation. These effects of protease inhibitors on basal progesterone production may be unrelated to angiotensin processing, and due to effects on the steroidogenic machinery. However, other studies have investigated ACE inhibition and ovarian steroidogenesis, and reported various effects. Inhibition of ACE activity in the frog ovary enhanced estradiol and progesterone production (4); in a hyperstimulated rabbit model, progesterone was unaffected and estradiol production was decreased (59); and in PMSG-treated rats, ACE inhibition by captopril infusion reduced serum levels of progesterone and enhanced estradiol output (2). The varying physiologic responses to ACE inhibition may be due to alternative angiotensinogen processing pathways in different species, but a further complicating factor is likely to be gonadotrophin treatment. Previous studies identified an interaction between LH and the OVRAS. LH stimulates bovine thecal cell production and secretion of renin/prorenin through a cyclic AMPdependent mechanism (6), and in vivo, gonadotrophin stimulation leads to enhanced ovarian Ang II levels in the bovine and human (1, 39). Whether this effect is a direct response to gonadotrophin or a secondary event is unclear. Bovine luteal ACE has been located to the endothelial cell population, and


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its activity is upregulated by a combination of estradiol and vascular endothelial growth factor (VEGF), but not estradiol alone (25, 33). However, the testicular ACE isoform (ACET) is regulated by gonadotrophins by a cAMP response element on the ACE gene (30, 75). We found an LH-dependent increase in ACE activity in cultured rat luteal cells that may be mediated by the LH second messenger cAMP. It is possible that enhanced ACE activity occurred in a population of endothelial cells as a secondary event to LH-stimulated endocrine cells. However, the luteal cells are purified by densitygradient sedimentation, and are principally steroidogenic cells (73), suggesting a direct effect of LH on luteal ACE. The hypothesis of LH-regulated luteal ACE activity is also supported by a report that inhibition of ACE in women during normal menstrual cycles is without effect on serum steroid levels, but in gonadotrophin-stimulated women during the luteal phase of IVF cycles, ACE inhibition reduces serum levels of progesterone and elevates estradiol (46). In our studies, progesterone output was unaffected by quinapril under basal conditions, but in LH-stimulated cells, an inhibitory effect was indicated when quinapril was included with ZPP. Taken together, inhibition of ACE activity may principally have steroidogenic effects in gonadotrophin-stimulated cells, but it remains to be determined whether enhanced ACE activity and corresponding Ang II production play a role in LHstimulated steroidogenesis. LH-stimulated progesterone production was also inhibited by ZPP, PCMS and PMSF. PCMS and PMSF had the most profound inhibitory effects on progesterone production, but only PCMS affected accumulation of Ang II and Ang-(1-7). We did not test the effect of exogenous Ang II or Ang-(1-7) with PCMS, but these peptides had no significant effect on progesterone production when added by themselves. The actions of PCMS and PMSF may be related to cholesterol transport (see below), and further studies are required to identify if their anti-steroidogenic sites of action include angiotensin processing.


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Ang-(1-7) stimulates progesterone output in perfused rat ovaries from gonadotrophin primed animals (13), but we found no effect of exogenous Ang II or Ang-(1-7) on progesterone production in luteal cells. Furthermore, ZPP that inhibits PEP, a mediator of Ang-(1-7) production, had differential effects on progesterone that related to gonadotrophin stimulation, since basal progesterone was enhanced and LH-stimulated progesterone was inhibited by ZPP. These different responses may be specific for tissue, or due to hormonal pre-treatment. We do not know whether LH regulates Ang-(17) accumulation, but luteal ACE activity was enhanced by LH, and since Ang-(1-7) production may occur via ACE and PEP activity, elevated Ang II may drive elevated Ang-(1-7) production. We found that quinapril plus ZPP reduced accumulation of Ang-(1-7), while a trend of reduced Ang II and Ang III accumulation did not reach statistical significance. In LH-stimulated cells, quinapril plus ZPP significantly reduced progesterone production to a greater extent than did either quinapril or ZPP alone, but this effect was not reversed by exogenous Ang II or Ang-(1-7). It is possible that the inhibitory action of quinapril plus ZPP are due to direct effects on the steroidogenic machinery, or alternatively, intracellular angiotensin processing may modulate steroidogenesis. In support of this latter hypothesis, intracellular generation of Ang II, and intracellular effects mediated by nuclear and cytosolic Ang II binding sites have been described in different cell types (17, 24, 26, 31, 71). Indeed in the present studies, immunofluorescence and confocal microscopy shows intracellular accumulation of Ang II within cytoplasm and nuclei. Therefore, we tested the effects of agonists and antagonists in permeabilized luteal cells that allow penetration of extracellular molecules to the intracellular milieu. In this paradigm neither Ang II, nor Ang-(1-7) significantly affected progesterone compared to control levels, although luteal cells incubated with Ang II produced significantly more progesterone than did cells incubated with Ang-(1-7). Furthermore, progesterone production was significantly reduced by losartan, and this effect was reversed when Ang II was included with the receptor antagonist. The AT2


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antagonist, PD123319 had no effect on steroidogenesis in intact or permeabilized cells. These data suggest that intracellular levels of Ang II can modulate steroidogenesis via AT1 receptors. Investigation of the mechanism of action of the protease inhibitors included adenylate cyclase activity that mediates LH-stimulation of steroidogenesis. Quinapril, ZPP and PMSF did not affect cAMP levels in either basal or LH-stimulated paradigms, but in contrast, PCMS did inhibit LHstimulated cAMP accumulation. This effect was not due to enhanced degradation of cAMP by phosphodiesterase activity, because the phosphodiesterase inhibitor, IBMX did not reverse PCMSinduced effects. We cannot rule out that a protease-dependent requirement for LH binding to its receptor (22) is a factor, or that PCMS may have a direct effect on adenylate cyclase activity (42). However, PCMS also exerted effects on steroidogenesis at a site post-adenylate cyclase because dbcAMP-stimulated progesterone was blocked by PCMS. Basal progesterone was largely unaffected by PMSF and PCMS, except for a high concentration of PCMS, but a more pronounced inhibition of progesterone production occurred in LH-stimulated cells that could be related to cholesterol transport. Investigation of the post-adenylate cyclase site of action utilized 22OH-cholesterol that permeates mitochondria independently of transport mechanisms. PMSF-dependent inhibition of steroidogenesis was fully reversed, while PCMS-dependent inhibition was mostly restored by 22OH-cholesterol. Therefore, PMSF and PCMS may affect cholesterol transport to the mitochondria and/or P450scc at sites including: sterol carrier protein 2 (SCP2) that regulates cholesterol transport from the lipid droplet to the mitochondria; steroidogenesis activator polypeptide; and steroid acute regulatory peptide (StAR) (49, 68, 74). However, SCP2 is slowly turned over (58), and therefore seems unlikely to be effected by the acute treatment in the present studies. On the other hand, StAR is rapidly synthesized in response to LH and cAMP, and is a key protein for transport of cholesterol from the outer to the inner mitochondrial membrane where the P450scc resides (10, 56). During its import to the inner


22

membrane, StAR is proteolytically degraded from a 37 kDa precursor to a 30 kDa protein (55, 67) and may be sensitive to PCMS and PMSF. Indeed, in adrenal, and Leydig cells, PMSF blocks stimulated steroidogenesis, at least in part, by inhibition of turnover of the 37 kDa StAR precursor (44). It is unknown, whether quinapril or ZPP affect the 37 kDa StAR precursor but their combined inhibitory effect was reversed by 20OH-cholesterol, suggesting an inhibitory site of action before P450scc. Better understanding of the complexity of the OVRAS may allow new diagnostic and treatment strategies. In beginning to examine such complexities, we conclude from these studies that angiotensin processing in luteal cells includes the formation of Ang II, Ang-(1-7) and Ang III. Exogenous Ang II or Ang-(1-7) do not affect the acute production of progesterone in either basal or LH-stimulated paradigms, however inhibitors of ACE and PEP cause inhibition of LH-stimulated steroidogenesis. This effect is not mediated by adenylate cyclase activity, and includes an inhibitory site of action before P450scc. We detected accumulation of Ang II in cytoplasm and nuclei of luteal cells that may modulate progesterone production, because in permeabilized luteal cells, progesterone production was reduced by the AT1 antagonist losartan, and this effect was reversed by Ang II. The permeabilized cell model did not detect an effect of intracellular Ang-(1-7) on steroidogenesis, but a cytosolic compartment for PEP that hydrolyses Ang I to Ang-(1-7) (64, 80) suggests intracellular generation of the hepta-peptide. Further studies are necessary to confirm a regulatory role for intracellular angiotensin peptides in steroidogenesis, and at what site of the steroidogenic pathway quinapril and ZPP may act.


23

ACKNOWLEDGMENTS We gratefully acknowledge DuPont Merk for donating Losartan, and to Parke-Davis for providing Quinapril and PD123319. This work was supported in part by NIH Grant HD-22970, and by the Department of Pathology, Women & Infants Hospital, Providence, RI 02905. Current address: Gabor Nemeth M.D., Department of Obstetrics and Gynecology, Albert Szent-Gyorgi University SzegedSemmelweis 1, Hungary; Yuji Yamada M.D. Department of Obstetrics and Gynecology, Nihon University School of Medicine, 30-1 Ohyaguchi Kamimachi, Itabashiku, Tokyo, Japan.


24

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Table 1 The effects of protease inhibitors on accumulation of angiotensin peptides by luteal cells. Levels of Ang-(1-7), Ang II and Ang III in control incubations that contained no inhibitors were set at 100%, and the effects of the inhibitors (quinapril, ZPP, PCMS and PMSF) were expressed as a percentage of the controls. Data are the mean ± SEM. Statistically significant differences from controls are indicated by an asterisk (* p < 0.05). Ang(1-7)

Ang II

Ang III

(% of control)

(% of control)

(% of control)

PMSF (n=4)

102.4 ± 14.8

94.9 ± 8.9

106.7 ± 13.9

PCMS (n=8)

59.9 ± 12.6*

67.2 ± 6.5*

30.1 ± 6.0*

Quinapril (n=5)

94.5 ± 14.4

80.4 ± 9.3

49.2 ± 10.0*

ZPP (n=5)

91.3 ± 5.5

135.9 ± 14.8*

90.7 ± 14.5

Quinapril + ZPP (n= 5)

56.8 ± 3.6 *

89.3 ± 7.4

83.3 ± 9.2


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Table 2 Progesterone production by rat luteal cells that were preincubated with ZPP plus quinapril (100 µM each) for 60 minutes, then subsequently incubated with 22OH-cholesterol ( 20 µg/ml) and LH (100 ng/ml) for 60 minutes. Data are the mean + SEM, n=4.

Control

ZPP+Quinapril

(ng/105 cells)

(ng/105 cells)

46.9 + 2.9

52.1 + 2.7


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Table 3 Effect of Ang II on LH-stimulated progesterone production. Luteal cells were incubated with LH (100 ng/ml) for 1 h in the absence or presence of increasing concentration of Ang II or Ang-(1-7). Data are the mean ± sem of n repeated experiments, and expressed as the percentage of progesterone produced by control incubations that contained LH alone (Controls = 100 %). (n/a = not assayed)

Concentration

Ang II

Ang-(1-7)

1 nM

N/A

94.1 ± 2.3, n = 5

10 nM

89.6 ± 8.4, n = 5

91.5 ± 2.1, n = 5

100 nM

94.5 ± 7.4, n = 5

89.8 ± 2.7, n = 5

1 µM

89.0 ± 7.5, n = 6

89.7 ± 2.9, n = 5


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FIGURE LEGENDS Figure 1: Immunolabeled Ang II in Luteal cells Rat luteal cells were isolated and processed for immunolabeling with anti-Ang II and DAB, as described in the Methods Section. Images were obtained under DIC, and Panel A shows the dark staining of immunolabeled cells, while Panel B shows the absence of dark staining when the primary antibody was omitted. (Bar represents 20 µm).

Figure 2: Distribution of Ang II Immunolabeling in Rat Luteal cells. Rat luteal cells were cultured for 24 hour, fixed and processed for immunohistochemistry and imaged by confocal fluorescence microscopy. Images were obtained with FITC and TRITC filters and a composite of six stacked images, taken at 1 µm sections were used to construct the images. In the left panel Ang II is labeled throughout the luteal cells with anti-Ang II antibody and fluorescein-labeled anti-rabbit IGG. The center panel identifies nuclei stained with propridium iodide, and the right panel is an overlay of FITC and TRITC images, showing cytoplasmic and nuclear accumulation of Ang II staining. (Bar represents 20 µm).

Figure 3: HPLC Elution Profiles. Panel A is an elution profile for standards of Ang-(1-7), Ang II and Ang III (each 200 pmoles). Panel B is a chromatogram showing the elution profile of luteal cell homogenate incubated with angiotensinogen (25 µM).

Figure 4: Dose-response effects of quinapril, ZPP, PCMS and PMSF on progesterone production. Luteal cells were preincubated with protease inhibitors (PMSF and PCMS for 30 minutes; quinapril and ZPP for 60 minutes), and subsequently further incubated in the absence (upper panel) or presence


33

of LH (100 ng/ml) for 60 minutes (lower panel). Statistical analysis by Two Way ANOVA was conducted on arcsine transformed data; however the figure depicts non-transformed data, expressed as a % of control incubations in the absence of inhibitor (mean ± SEM of a minimum of three repeated experiments for each inhibitor). Controls = 100 %. (Two way ANOVA and Student-Newman-Keuls post-hoc test revealed significant differences from control (p < 0.05) that are identified by an asterisk *. Means identified “a “versus “b”, p < 0.05).

Figure 5: Dose-response effects of quinapril, ZPP, PCMS and PMSF on cAMP accumulation. Luteal cells were preincubated with protease inhibitors (PMSF, PCMS, quinapril, or ZPP) as described in Figure 4. Cells were further incubated in the absence (upper panel) or presence of LH (100 ng/ml) for 60 minutes (lower panel). Statistical analysis by Two Way ANOVA was conducted on arcsine transformed data; however the figure depicts non-transformed data, expressed as a % of control incubations in the absence of inhibitor (mean ± SEM of a minimum of three repeated experiments for each inhibitor). Controls = 100 %. (Two way ANOVA and Student-Newman-Keuls post hoc test revealed significant differences from control (p < 0.05) that are identified by an asterisk.

Figure 6: Effect of PCMS on IBMX- and dbcAMP-incubated luteal cells. The upper panel shows accumulated cAMP in cells preincubated with PCMS as described in Figure 4, and subsequently incubated for 60 minutes in the absence or presence of LH (100ng/ml) with and without IBMX (200 µM). The lower panel shows progesterone production by cells preincubated with PCMS, followed by incubation with PCMS and LH (100 ng/ml) with IBMX (200 µM), or dbcAMP (1 mM) for 60 minutes. Data from a representative experiment are shown, and are the mean ± SEM (n=4). Two Way ANOVA


34

/ Student-Newman-Keuls revealed significant differences from control means (p < 0.05), identified with an asterisk.

Figure 7: Reversal of inhibitory effects of PCMS and PMSF on progesterone production. Rat luteal cells were preincubated for 30 minutes in the absence or presence of PCMS (100 µM) (upper panel) or PMSF (100 µM) (lower panel). Subsequently, cells were further incubated for 60 minutes in the presence of LH (100 ng/ml), and in the absence or presence of 22OH-cholesterol (20 µg/ml). Data from representative experiments are shown, and are the mean ± SEM, n=4. Means indexed with different letters are significantly different, ANOVA p < 0.05.

Figure 8: Upper Panel shows the combined effects of ZPP and quinapril on LH-stimulated progesterone production. Luteal cells were incubated in the presence of increasing concentrations of ZPP with or without quinapril (100 µM) for 60 minutes, and then incubated for 60 minutes with LH (100ng/ml). In the lower panel, cells were incubated with quinapril and ZPP (each 100 µM) as described for the upper panel, and Ang II (1 µM) or Ang-(1-7) (1 µM) were added at the same time as LH, and incubated for 60 minutes. Data are expressed as a % of the progesterone produced by cells incubated with LH alone (100 %), and are the mean ± SEM of 3 – 5 experiments. Statistical analysis for upper panel: Two Way ANOVA of arcsin transformed data showed significant differences among treatments (p < 0.05), and paired t-test comparison of ZPP against ZPP + quinapril, p < 0.005. Lower panel: ANOVA, * p < 0.05 vs control.


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Figure 9 ACE activity in Luteal Cells. Rat luteal cells were incubated for 24 hours in the absence or presence of LH (100 ng/ml). Subsequently, ACE activity was determined using ABGP as described in the Methodology section. Data are corrected for substrate blanks, and are expressed as % fluorescence of control incubations. Quinapril (100 µM) was included where indicated. Data from one experiment are shown, and are the mean ± SEM, n=4. Means indexed with different letters are significantly different, p < 0.05.

Figure 10 Effect of modulation of intracellular angiotensin peptides on progesterone production. Luteal cells were permeabilized as described in the Methods section, and incubated for 60 minutes with LH (100 ng/ml) and: (upper panel) Ang II (1 µM) and Ang-(1-7) (1 µM); or (lower panel) losartan, (1 µM), PD123319 (1 µM), and losartan plus Ang II (each 1 µM). Data are expressed as a % of control incubation with LH alone (100 %) and are the mean ± SEM, n=3 experiments. ANOVA: * p < 0.05 vs. Control. Means indexed with different letters are significantly different, p < 0.05.

Luteal Angiotensins and Steroidogenesis Figure 1

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