Rapid plasma membrane changes in superoxide radical formation, fluidity, and phospholipase A2 activity in the corpus luteum of the rat during induction of luteolysis.

0013-7227/91/1286-2992$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 6 Printed in U.S.A.

Rapid Plasma Membrane Changes in Superoxide Radical Formation, Fluidity, and Phospholipase A2 Activity in the Corpus Luteum of the Rat During Induction of Luteolysis* MASAAKI SAW ADA AND JOHN C. CARLSON Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl

ABSTRACT. Early luteolytic changes in the plasma membrane of luteal cells were examined in the rat. Treatment with prostaglandin F2cv in vivo caused a rapid transient increase in superoxide radical formation and a decrease in fluidity in plasma membrane samples prepared from luteinized rat ovaries. These alterations preceded detection of a significant fall in plasma progesterone concentration. The rise in superoxide radical was not accompanied by changes in activities of free radical scavenging enzymes. Within the first hour of prostaglandin treatment,

there was also a significant increase in the activity of phospholipase A2 and ATP-dependent calcium uptake in the membrane samples. These experiments indicate that one of the initial sites affected by the luteolytic process appears to be the plasma membrane. The changes include a transient rise in production of superoxide radicals, which may cause membrane changes that are responsible for disrupting corpus luteum function in the rat. (Endocrinology 128: 2992-2998, 1991)

R

EGRESSION of the corpus luteum (CL) is characterized by functional and structural alterations which result in the loss of steroidogenesis by the luteal cell. Although a number of cellular changes associated with regression have been described, the sequence of modifications responsible for the loss in progesterone secretion is unknown. The acute effects of prostaglandin F2« (PGF2«) treatment have been examined during in vivo and in vitro experiments. In the former studies, sc injection of PGF2« results in significant decreases in serum progesterone, uptake of gonadotropic hormone (1), and plasma membrane fluidity (2) within 1 h of administration. Exposure of luteal cells to PGF2a in vitro produces a significant increase in hydrolysis of phosphoinositides (3, 4), elevation of cytosolic Ca2+ concentrations (4), and inhibition of gonadotropin-stimulated cAMP formation (5). Recently Behrman and Preston (6) reported that treatment of isolated rat luteal cells with H2O2 caused rapid inhibition of LH-stimulated cAMP accumulation and progesterone production within 5 min. There is evidence that free radicals may be involved in CL regression. Free radicals are atoms or molecules

that contain one or more unpaired electrons (7), and they can damage cells due to their reactive nature (8). The superoxide radical (SOR), which is a by-product of normal metabolic activity, is continuously produced by mitochondria. This radical also is generated by the plasma membrane of neutrophils, as they produce a respiratory burst to kill invading organism (9). In addition, recent studies indicate that this oxyradical is produced by the plasma membrane of rat luteal cells during luteolysis (10). Free radicals induce lipid peroxidation (11), which increases during CL regression (12). Lipid peroxidation leads to further membrane breakdown including cross-linking in proteins and lipids, formation of the gel-phase lipid (13), which occurs in regressing CL (14), and stimulation of prostaglandin biosynthesis (15). For removal, cells degrade SOR to H2O2 by superoxide dismutase (SOD), and the peroxide is converted to H2O by catalase. However, H2O2 can react with SOR in the presence of iron to form hydroxyl radicals by the HaberWeiss reaction (16). Hydroxyl radicals are more destructive than SORs or H2O2. As a result of the above information, we decided to examine if SOR formation coincides with the interruption in progesterone secretion and if such an alteration is associated with acute plasma membrane modifications.

Received December 31, 1990. Address all correspondence and requests for reprints to: Dr. J. C. Carlson, Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. *This work was supported by the Medical Research Council of Canada.

Materials and Methods Animals Immature (23-25 day old) rats were superovulated with PMSG and human (h) CG as described previously (10). To

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PLASMA MEMBRANE CHANGES DURING LUTEOLYSIS initiate regression, a total of 500 ng PGF2n (Dinoprost Tromethamine, Tuco Products Co., Orangeville, Ontario, Canada) were injected sc at eight separate sites along the dorsal surface 7 days after hCG. Control rats were injected in a similar manner with saline. Before killing the animals by cervical dislocation, blood samples were collected from ether-anesthetized rats for plasma progesterone measurements by RIA (17). Experiments were replicated a minimum of five times with separate groups of rats (two animals) in each replicate. Plasma membrane preparation Ovaries were removed at various times after PGF2n administration, and plasma membrane samples were prepared using a modified dextran-polyethylene glycol, two-phase system (10). In the present study, ovaries from two rats were homogenized as described and centrifuged (700 x g, 10 min) to remove cellular debris. The supernatant was spun for 10 min at 3000 X g (4 C) in a fixed angle rotor. The supernatant from the second spin was discarded, and the pellet was removed and mixed with 25 ml dextran-polyethylene (1:1) containing 10~6 M ZnCl2 and vortexed vigorously. The mixture was spun for 10 min at 9000 X g (4 C) in a fixed angle rotor. The protein level of the membrane sample, located at the interface, was determined by using the bicinchoninic acid reagent according to the procedure described by Sorensen and Brodbeck (18). Marker enzymes The plasma membrane samples were assayed for marker enzymes to determine source and enrichment. Ouabain-sensitive K + /Na + ATPase was used as the plasma membrane marker (19), NADH cytochrome C reductase activity, for the endoplasmic reticulum marker (20), and succinate dehydrogenase activity (21), as the mitochondrial marker. The results indicated that enrichment of the plasma membrane was 10-fold (Table 1). Total phosphate was determined using the method described by Dittmer and Wells (22). Electron spin resonance (ESR) Levels of SOR in membrane samples were determined by measuring the height of the ESR signal produced by reaction of this radical with freshly prepared Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) (23, 24). The characteristic 4-peak spectrum (10, 24) was measured at 5 min, which corresponds TABLE 1. Activities of marker enzymes in plasma membrane samples Succinate NADH cytochrome (K+-Na+)ATPase dehydrogenase C reductase 0.10 ± 0.02 2.99 ± 0.19 6.62 ± 0.30

Specific activity Enrichment 0.77 ± 0.11 2.97 ± 0.19 10.08 ± 0.45 The specific activity of succinate dehydrogenase (mitochondrial marker) represents the micromolars of p-iodonitrotetrazolium violet reduced per mg protein/h. The specific activity of cytochrome C reductase (endoplasmic reticulum marker) refers to the micromolars of cytochrome C reduced per mg protein/h. The specific activity of (K+Na+)ATPase (plasma membrane marker) represents the micrograms of PO4 produced per mg protein/h. Enrichment equals the specific activity of the membrane sample per specific activity of homogenate. Each value represents the mean ± SE offivereplicates.

2993

to the time when a steady state was reached (25). The basic procedure followed that described previously in our laboratory (10, 26, 27) except that 150 \ig membrane protein were used for each sample. The concentration of CaCl2 was 0.5 mM. The spectrometer was calibrated using the xanthine oxidase/xanthine SOR generating system (28). To examine specificity, SOD was added to the membrane samples. The SOR signal was essentially eliminated upon exposure to this enzyme. Fluorescence polarization Plasma membrane fluidity changes were measured by fluorescence polarization using the probe trans-parinaric acid as described previously (29) with minor modifications. In the present study, the buffer contained 0.25 mM CaCl2, and the sample chambers were sealed under nitrogen to inhibit oxidation. Measurements were made in a spectrofluorimeter (SLM model 8000, SLM, Champaign, IL). Phospholipase A2 activity Phospholipase A2 activity in the plasma membrane samples was determined by the method of Petkova et al. (30). The samples (100 ng of protein) were suspended in 100 mM Tris/ HC1 buffer (pH 8.5) containing 5 mM CaCl2. The substrate, 170 nmol l-acyl-2-[14C]linoleoyl-sn-glycerophosphoethanolamine, was added to the samples and incubated for 20 min. The released radiolabeled arachidonic acid was determined by liquid scintillation counting after chloroform/methanol extraction (2:1, vol/vol), separation by TLC in a solvent system containing chloroform/isopropanol/triethylamine/methanol/0.25% KC1 (30:25:18:9:6, vol/vol) (30, 31), and elution from the silica gel. The specific activity was expressed as nanomoles of fatty acid released per min/mg of membrane protein. Lipid peroxide measurement Evidence of lipid peroxidation in the plasma membrane samples was obtained by the thiobarbituric acid test (32) as modified by Sawada and Carlson (12). The results were expressed as nanograms of malonaldehyde (bis[dimethyl acetal]; Aldrich, Milwaukee, WI) per mg of protein. Ca2+ uptake

The method of Minami and Penniston (33) was used to determine ATP-dependent Ca2+ uptake in the plasma membrane samples prepared at different times after PGF 2a treatment. The reaction was started by adding 1.5 X 105 cpm/nmole of 45CaCl2 (200 M0 to the samples (50 Mg) in 500 fi\ 50 mM-(iVTris[hydroxymethyl|methyl-2-aminoethanesulfonic acid)/triethanolamine buffer, pH 7.4, containing 0.25 M sucrose, 0.1 mM ouabain, 5.0 mM MgCl2, and 50 nM CaCl2 with and without 6.0 mM ATP. After incubation for 20 min at 37 C, the samples were centrifuged at 100,000 X g for 20 min. The supernatant was removed, and the pellet was mixed with liquid scintillation fluid and counted. ATP-dependent calcium uptake was calculated by subtracting radiolabeled uptake in the absence from that in the presence of ATP. Free radical scavenging enzymes The activities of free radical scavenging enzymes were determined spectrophotometrically in samples of ovarian homoge-

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2994

PLASMA MEMBRANE CHANGES DURING LUTEOLYSIS

nates (100-200 ng protein). SOD activity was obtained by calculating the amount of enzyme required for 50% inhibition of cytochrome C reduction (34). Activity is based on the increase in absorbance at 550 nm and is expressed as units of enzyme per mg protein. The activity of catalase was determined by measuring the decomposition of hydrogen peroxide (35) and corresponded to the change in absorbance at 245 nm/min • mg of homogenate protein. The activity of glutathione peroxidase was determined by measuring, at 340 nm, the micromoles of NADPH oxidized per min/mg of homogenate protein (36). Statistical analyses To test for significance, one-way analysis of variance followed by the posthoc Tukey test was used for comparison between control and regressing groups. For comparison within each experimental group, Duncan's Multiple Range test was used. A statistical program, LCS Stat-Lab V2.20 (LCS Inc., Montreal, Quebec, Canada), was used for the calculations.

Results Plasma progesterone level Multisite sc injection of PGF 2a resulted in a significant drop in plasma progesterone (P < 0.01) 40 min after administration, when compared with pretreatment levels in this group (Fig. 1). Also, when compared with the control group, plasma progesterone concentration was significantly lower at 20 min (P < 0.05). Levels of this steroid hormone continued to decline after PGF2« treatment. At 6 h, the plasma concentration was less than 50 ng/ml. ESR signal The possibility of a rapid increase in SOR levels after in vivo PGF 2a treatment was examined in the plasma

Endo•1991 Vol 128 • No 6

membrane samples. The results in Fig. 2 show a significant 3-fold elevation (P < 0.005) in samples prepared from ovaries removed 10 min after PGF2a injection. This SOR burst returned to pretreatment levels in samples prepared from ovaries removed 40 min after PGF2cv injection, and it remained unchanged for the duration of this 2-h study. No increase in the ESR signal occurred in membrane samples from superovulated controls or in samples from PGF2«-treated rats if the membranes were exposed to 30 ng SOD for 60 min at 39 C or if they were heated to 100 C for 15 min before ESR spectroscopy. Fluorescence polarization Fluorescence polarization measurements are shown in Fig. 3. As in the previous experiment, ovaries were removed at various times after PGF2cv treatment. There was a significant decrease (P < 0.01), shown by an increase in the polarization ratio, in the fluidity of plasma membrane samples shortly after PGF2tt treatment. The changes were reversible and they paralleled those observed for the SOR study, except that membrane perturbation lasted somewhat longer. At 60 min the polarization value was not significantly different than the pretreatment reading. Phospholipase A2 activity Changes in phospholipase A2 activity in the plasma membrane samples prepared from ovaries removed up to 2 h after PGF 2a treatment are shown in Fig. 4. Although the activity was higher in many of the samples from PGF2a-treated rats than in control samples, the difference was significant only at 20 and 60 min (P < 0.5).

400

Control

T

3.0

350 60

a o

i* 4) n «

300

O PGF2a Treated

250 -

# Control

on 200 o

150 100 0.0

20

40

60

BO

100

120

Time (Min) FIG. 1. Plasma progesterone levels in PGF2(V-treated and control rats. The difference in plasma progesterone concentration between the two groups was significant at 20 min (P < 0.05). Each value represents the mean ± SE of eight replicates.

40

60

80

100

120

Time (min) FIG. 2. SOR production in plasma membrane samples prepared from control and PGF2a-treated rats. SOR levels were measured under steady state conditions, which occurred 5 min after warming samples to 37 C. A significant rise (P < 0.05) in SOR level appeared 10 min after PGF2n treatment. Each value represents the mean ± SE of five replicates.



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TABLE 2. Lipid peroxidation in plasma membrane samples

0.23 -i

Time (min)

0.22 -

PGF2o-treated (ng)

0 5 10 20 40 60 120

0.21 -

0.20 -

9.64 ± 9.64 ± 10.01 ± 11.87 ± 11.87 ± 15.95 ± 20.39" ±

Control (ng) 12.24 ± 5.00 11.87 ± 3.87 11.87 ± 3.87 9.64 ± 3.23 9.64 ± 3.23 10.01 ± 4.56 11.88 ± 3.87

3.23 3.23 4.56 3.87 3.87 2.72 3.52

Lipid peroxidation was determined spectrophotometrically by comparing the amount of thiobarbituric reactive material in each sample to that produced by malonaldehyde. Each value represents the mean ± SE of six replicates. 0 Significant at P < 0.06.

0.19

0.18

2.5 -i 20

40

60

60

100

120

Time (min) FIG. 3. Changes in plasma membrane fluidity after induction of luteolysis with PGF2n. Steady state fluorescence polarization measurements were performed after incubating samples for 90 min at 37 C in buffer containing 0.25 mM CaCl2. The fluidity decrease was significant (P < 0.05) in samples collected 10 min after PGF2a treatment. Each value represents the mean ± SE of six replicates. 4.0 T

20

40

60

60

100

120

Time (min) FIG. 5. ATP-dependent Ca2+ uptake in plasma membrane samples from control and PGF2n- treated rats. Uptake is expressed in nanomoles per mg of protein. Samples were incubated at 37 C for 20 min after addition of 45CaCl2. The increase in the treated group was significant at 60 and 120 min (P < 0.05). Each value represents the mean ± SE of five replicates.

Control

Ca2+ uptake

2.6 20

40

60

60

100

120

Time (min) FIG. 4. Phospholipase A2 activity in nanomoles of arachidonic acid released per min/mg of membrane protein in plasma membrane samples from control and PGF2a-treated rats. Samples were incubated 20 min at 37 C in buffer containing 0.25 mM CaCl2. The activity was significantly greater in samples prepared from ovaries removed 20 and 60 min after PGF 2 Q treatment when compared with the corresponding controls (P < 0.05). Each value represents the mean ± SE of five replicates.

Lipid peroxidation One of the primary effects of free radical damage is lipid peroxidation. In the present study, the rate of lipid peroxidation began to increase and approached significance (P < 0.06) 2 h after PGF2rt treatment (Table 2).

Ca2+ uptake increased significantly in plasma membrane samples prepared from ovaries of PGF2«-treated rats (Fig. 5). The rise was significant at 60 and 120 min (P < 0.05) within the treatment group or between control and treated groups (P < 0.05). Addition of the Ca2+ ionophore, A23187, at the end of the 20-min incubation caused complete release of Ca2+. Enzyme profile Activities of the free radical scavenging enzymes, SOD, catalase, and glutathione peroxidase, and the activity of K + /Na + ATPase were examined to see if they change immediately after PGF2« induction of luteolysis. No change in activities were observed between samples from control and treated rats within 2 h of PGF2w injection.


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Discussion It is clear that plasma membrane changes associated with functional luteolysis in the rat begin within minutes of PGF2« treatment in vivo. The modifications include transient alterations in SOR formation and membrane fluidity in samples collected 10 min after injection. A significant fall in plasma progesterone was first detected at 20 min. The sequence of these changes strongly suggest that they are involved in the initial interruption in progesterone secretion. Oxyradicals, such as SOR, are toxic to cells. These agents, which frequently appear in fluxes, induce molecular breakdown, disruption of cell function, and death (37). The targets include enzymes, nucleotides, and membranes (11, 16). Evidence of membrane damage appeared in the fluorescence polarization experiment, in which a decrease in fluidity was recorded. Oxidative attack results in lipid peroxidation, as noted in the present and in previous studies (12), protein polymerization and cross-linking, which alter fluidity characteristics (11). Fluidity changes affect a number of membrane functions including receptor binding (38), carrier-mediated transport (39), and enzyme activity such as adenyl cyclase (40). In the present study, one site that the rapid burst in SOR formation appears to be directed at is the plasma membrane. The resulting perturbation, as evidenced by the concurrent change in fluidity, may be responsible for the loss in progesterone secretion. Another site that may be considered is the endoplasmic reticulum. Membranes from the organelle, which participate in steroidogenesis, were also present in the sample preparation. Generation of the SOR burst appears to be enzymatic as indicated in previous studies (9,10) and in the present investigation by the observation that the increase was temperature sensitive. Alterations associated with the plasma membrane samples, which required 2-4 h to prepare, were minimized by maintaining samples at 4 C in an enriched nitrogen atmosphere. Reversibility of the above changes suggest that the observed events are representative of in vivo alterations. Blockade by SOD of the expected SOR increases indicates that the method of detection, ESR spectroscopy with Tiron as the spin trap, is specific for this oxyradical. The temporary increase in SOR formation and associated membrane changes also may be responsible for stimulation of phospholipase A2, which causes phospholipid deesterification and free fatty acid release. Membrane perturbation activates phospholipase A2 (41), and activation during luteolysis has been reported (2, 29). In addition, the products of deesterification, namely lysophospholipids and free fatty acids, such as arachidonic acid, may work directly to perturb the plasma membranes of luteal cells as demonstrated in an earlier study (42).

Endo«1991 Voll28«No6

Free arachidonate also may enter metabolic pathways that are involved in luteolysis. It is metabolized to PGF2«, which could function locally to reinforce the luteolytic signal. Also, it has been shown that this fatty acid stimulates SOR-generating enzymes in human neutrophils (9). Evidence that phospholipase A2 participates in SOR formation in regressing rat CL was obtained previously (10). Thus, deesterification of fatty acids may provide substrates for several destructive pathways in the luteal cell. Ca2+ appears to be involved in the luteolytic mechanism (43). PGF 2a activates phospholipase C in the luteal cell (3) and results in Ca2+ release (4); this divalent cation is also associated with SOR formation in human neutrophils (44). In the present study, Ca2+ uptake increased significantly in samples removed 60 min after PGF2« treatment. Uptake was temperature-, ATP-, and Mg2+dependent, and Ca2+ was released by addition of A23187 to the plasma membrane samples. These features characterize a Ca2+ transport enzyme, which has been described previously in luteal cell membranes (33). The results suggest that a homeostatic mechanism is working to reduce the toxic effect of high intracellular Ca2+ levels that occur during luteolysis. Arachidonic acid stimulation of Ca2+ efflux in macrophages has been described (45). Although the sites of luteolytic changes are unknown, it seems possible that free radical attack during regression may be localized. Site-specific attack by oxyradicals in cellular membranes has been proposed (16). During induction of regression, PGF2a treatment causes a substantial decrease in hCG binding capacity in samples prepared from rat ovaries (46, 47) without a corresponding decrease in Na + /K + ATPase or 5'-nucleotidase activity, as observed previously (47). Also, we observed no change in Na + /K + ATPase activity in the present study. Responsible for maintaining ion gradients, this enzyme consumes a considerable portion of the cell's energy (48) and it is believed to be sensitive to membrane fluidity changes (49). Additional evidence of localized disruption appears in the fluorescence polarization experiment. The probe, trarcs-parinaric acid, preferentially partitions into less fluid regions (50); similar studies in which plasma membrane samples from regressing CL were examined with the fluorescence probe, diphenyl hexatriene, failed to show parallel fluidity changes (14), presumably because of differential accessability to membrane domains. The inability to detect changes in free radical scavenging enzyme activities also indicates that in the rat the initial luteolytic changes do not cause disruption in all parts of the cell. However, it is possible that other cellular sites not examined in the present study may be affected in the early stages of regression. The above experiments indicate that stimulation of



SOR formation by PGF2« is one of its earliest events in the luteolytic process.. Within our plasma membrane samples, it appears that this burst induces a series of changes that stimulate lipolysis and substrate generation for continued membrane disruption. Initially, the luteolytic alterations seem to be localized and responsible primarily for interrupting the pathway involved in regulating progesterone secretion.

Acknowledgments The authors would like to thank Mr. H. Sawada for his assistance in developing the data acquisition and support programs for this study.

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In vivo generation of hydrogen peroxide in the rat corpus luteum during luteolysis.

Stimulation of phospholipase A2 by xanthine oxidase in the rat corpus luteum.

Lymphatic involvement in the disappearance of steroidogenic cells from the corpus luteum during luteolysis.

Histological analysis of arteriovenous anastomosis-like vessels established in the corpus luteum of cows during luteolysis.

Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane.

Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Membrane structure, toxins and phospholipase A2 activity.

The size of the corpus luteum during pseudopregnancy and pregnancy in the rat.

Galectin-3 contributes to luteolysis by binding to Beta 1 integrin in the bovine corpus luteum.

Role of intraluteal estrogen in the regulation of the rat corpus luteum during pregnancy.

Synaptosomal membrane fluidity, lipid peroxidation and superoxide dismutase activity in the brain of amygdala-kindled rats.

A pH-dependent phospholipase A2 contributes to loss of plasma membrane integrity during chemical hypoxia in rat hepatocytes.

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Corpus luteum susceptibility to prostaglandin F2 alpha (PGF2 alpha) luteolysis in hysterectomized prepuberal and mature gilts.

Ontogeny of cytosolic phospholipase A2 activity in rat brain.

Changes in concentration of prostaglandin F and steroids in human corpora lutea in relation to growth of the corpus luteum and luteolysis.

Induction of group II-like phospholipase A2 by lipopolysaccharide in the liver of BCG-primed rat.

Cytological observations of dissociated rat corpus luteum.

The induction of a secondary corpus luteum on day 12 post-ovulation can delay the time of luteolysis in high-producing Holstein cows.

The beta adrenergic receptors of rat corpus luteum membranes..

Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane.

The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis, and the phagocytosis of luteal cells.

Net activity of phospholipase A2 in brain and the lack of stimulation of the phospholipase A2-acylation stem.