Renal epoxyeicosatrienoic acid synthesis during pregnancy - 《American Journal of Physiology》美国生理学杂志干细胞之家



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Department of Physiology, Center for Biotechnology and Genomic Medicine, and Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia1 O2 X( n# E& Q3 a- \' t

ABSTRACT: V% H8 Y& C* s/ u5 C# i9 R: V8 F

Epoxyeicosatrienoic acids (EETs), which belong to cytochrome P-450 (CYP)-derived eicosanoids, have been implicated to vasodilate renal arterioles, inhibit sodium transport in the nephron, and regulate blood pressure in several animal models. Because pregnancy is associated with changes of blood pressure, the aim of this study was to examine whether renal EET synthesis is altered and whether EETs are involved in blood pressure regulation during pregnancy in rats. Renal microsomal epoxygenase activity increased by 47, 97, and 63% on days 6, 12, and 19 of gestation, respectively. The elevation of epoxygenase activity during pregnancy was associated with an increase in CYP2C11, CYP2C23, and CYP2J2 protein expression on days 6, 12, and 19 of gestation. Moreover, immunohistochemical analysis showed that renal tubular CYP2C11, CYP2C23, and CYP2J2 expression was significantly increased in pregnant rats on days 6, 12, and 19 of gestation. Administration of 6-(2-propargyloxyphenyl)hexanoic acid (PPOH), a selective epoxygenase inhibitor, caused a dose-dependent inhibition of microsomal expoxygenase activity without a significant effect on -hydroxylase activity in female rats. Interestingly, administration of PPOH (20 mg﹞kg–1﹞day–1 for 4 days starting on day 15 of pregnancy) increased blood pressure by 21 mmHg and caused a significant decrease in the body weight of fetal pups (1.3 ± 0.08 g in control vs. 1.1 ± 0.06 g in PPOH). Moreover, PPOH treatment significantly decreased renal microsomal epoxygenase activity and the expression of CYP2C11, CYP2C23, and CYP2J in pregnant rats. This study demonstrates that EET synthesis in the kidney is elevated during pregnancy, and CYP2C11, 2C23, and CYP2J2 are responsible for the change of renal EET synthesis. The inhibition results demonstrate that the downregulation of renal epoxygenase activity by PPOH causes hypertension in pregnant rats. This study suggests that EETs may contribute to the control of blood pressure during pregnancy.
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blood pressure; cytochrome P-450; epoxyeicosatrienoic acids8 q$ D# C" a  O) t6 e! L
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CYTOCHROME P-450 (CYP) enzymes constitute a major metabolic pathway for arachidonic acid in the rat kidney. In the presence of NADPH and oxygen, arachidonic acid is oxidized by the CYP enzyme system into four epoxyeicosatrienoic acids (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET). These EET molecules can be further hydrolyzed by epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acid (DHET). Studies have demonstrated that EETs play a significant role in the regulation of renal function by affecting tubular ion transport and by causing vasodilation (7, 17, 19). EET synthesis can be carried out by several CYP isoforms from the CYP1A, -2B, -2C, -2D, -2E, -2J, and -4A families (19). Although many CYP enzymes can carry out the epoxidation of arachidonic acid, several investigators have demonstrated that in the kidney, EET synthesis is primarily catalyzed by the CYP2C and CYP2J isoforms (3, 11, 18). On the basis of renal epoxygenase profiles and antibody inhibitory studies, it has been established that CYP2C23 is the predominant arachidonic acid epoxygenase in the rat kidney (6). Recent studies have shown that CYP2J proteins are expressed in the rat kidney, and the expression of CYP2J isoforms is increased in spontaneously hypertensive rats (15, 25).' b$ s+ Q- r3 _, i1 k" o! `/ o+ [8 {

Normal pregnancy in humans and rats is associated with increases in the glomerular filtration rate and renal blood flow (14) along with significant decreases in arterial pressure and total peripheral resistance (1, 12). EETs possess biological effects that can potentially contribute to these physiological changes during pregnancy. These biological effects include inhibition of ion transport along the nephron and vasodilation of renal arterioles (17, 19). In proximal tubules, EETs have been reported to inhibit Na -K -ATPase activity (21) and sodium transport (19). In renal microvessels, Imig et al. (9) have reported that 11,12-EET and 14,15-EET cause vasodilation. Moreover, 11,12-EET can activate vascular smooth muscle cell K  channels and has been proposed to be an endothelium-derived hyperpolarizing factor (4, 8). A role for EETs in the regulation of blood pressure has been further implicated in studies showing that stimulation of EET formation affects arterial pressure (7, 10). Moreover, arterial pressure of male mice null for soluble epoxide hydrolase was lower compared with wild-type mice (22), and the expression of renal soluble epoxide hydrolase was significantly increased in spontaneously hypertensive rats compared with Wistar-Kyoto rats (26). Although normal pregnancy in rats is associated with a significant decrease in arterial pressure (1, 14), the exact mechanisms mediating blood pressure changes during pregnancy are not fully understood. The initial aim of the present study was to test whether there is any alteration in EET synthesis and expression of CYP epoxygenases in the kidneys of pregnant rats. In addition, we studied the effect of 6-(2-propargyloxyyphenyl)hexanoic acid (PPOH), a mechanism-based inhibitor of CYP-derived EET synthesis, on blood pressure in pregnant rats during the third week of gestation. This study provides valuable information for evaluating the role of EETs in the regulation of blood pressure during pregnancy.4 a8 w& A0 D7 ?; M4 }# x: z2 y
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MATERIALS AND METHODS" S, `5 O) A8 p0 p$ C+ q+ E( N8 N5 c

Materials! q3 ~! _4 K6 s

[1-14C]arachidonic acid (56 mCi/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). PPOH was obtained from Cayman Chemical (Ann Arbor, MI). All solvents were HPLC grade.
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Animals
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All animals were purchased from Charles River Laboratories (Wilmington, MA). Experiments were conducted on pregnant (timed pregnancy) Sprague-Dawley rats (8 wk old) and age-controlled, virgin female Sprague-Dawley rats. Experimental protocols were approved by the Institutional Animal Care and Use Committee. Rats were maintained under controlled housing conditions of light and temperature and received standard laboratory chow and water.

Protocols to Evaluate Renal CYP2C11, CYP2C23, and CYP2J Expression and EET Production& R+ Y4 `9 u. ], j

The experiments were conducted in pregnant rats on the 6th, 12th, and 19th gestational day to represent early, middle, and late pregnancy. Virgin and pregnant rats (n = 6) were anesthetized with pentobarbital sodium (50 mg/kg ip) on the 6th, 12th, or 19th gestational day and instrumented with a polyethylene (PE-50) catheter in the femoral artery for blood pressure measurement. Mean arterial pressure (MAP) was measured using pressure transducers (model TRN050, Kent Scientific; Torrington, CT) coupled to a computer system (EMKA Technologies; Falls Church, VA). After blood pressure measurements were taken, the kidneys were removed, and the renal cortex was homogenized in buffer containing 100 mmol/l Tris﹞HCl and 1.15% KCl, pH 7.4. Homogenates were centrifuged at 10,000 g for 30 min. Microsomes were obtained by centrifugation of the supernatant at 100,000 g for 90 min and were resuspended in 0.25 mol/l sucrose buffer and stored at –80°C.% t- ?- T1 F) @: R8 k& _( b# L/ {  f( C

Arachidonic acid metabolism. Renal cortical microsomes (150 μg) isolated from pregnant and virgin rats were incubated with [1-14C]arachidonic acid (0.4 μCi, 7 nmol) and NADPH (1 mmol/l) in 0.3 ml potassium phosphate buffer (100 mmol/l, pH 7.4) containing 10 mmol/l MgCl2 for 30 min at 37°C. The reaction was terminated by acidification to pH 3.5–4.0 with 2 mol/l formic acid, and arachidonic acid metabolites were extracted with ethyl acetate. The ethyl acetate was evaporated under nitrogen, and the metabolites were resuspended in 50 μl methanol and subjected to reverse-phase HPLC using a 5-μm ODS-Hypersil column, 4.6 x 200 mm (Hewlett-Packard; Palo Alto, CA), and a linear gradient of acetonitrile-water-acetic acid ranging from 50:50:0.1 to 100:0:0.1 at a flow rate of 1 ml/min for 30 min. The elution profile of the radioactive products was monitored by a flow detector (In/us System; Tampa, FL). The identities of arachidonic acid metabolites [20-hydroxyeicosatetraenoic acid (HETE), DHETs, and EETs] were confirmed with authentic standards. The activity of the formation of these metabolites was estimated based on the specific activity of the added [1-14C]arachidonic acid and was expressed as picomoles per milligram of protein per minute.2 ]$ J: y, b0 F5 _9 q0 o9 U
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Western blot analysis. Renal microsomes (10 μg) from pregnant and control virgin rats were separated by electrophoresis on a 10 x 20-cm, 8% SDS-polyacrylamide gel at 25 mA/gel at 4oC for 18–20 h. The proteins were transferred electrophoretically to an enhanced chemiluminescence (ECL) membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 10 mmol/l Tris﹞HCl, 0.1% Tween 20, and 150 mmol/l NaCl for 90 min and then washed three times with TBS. The membranes were incubated for 10 h with goat anti-rat CYP2C11 (1:2,000, Gentest; Woburn, MA), rabbit anti-rat CYP2C23 antibody (1:5,000, a gift from Dr. J. H. Capdevila, Vanderbilt University, Nashville, TN), or rabbit anti-human CYP2J2 antibody (1:2,000, a gift from Dr. D. C. Zeldin, National Institute of Environmental Health Science, Research Triangle Park, NC) at room temperature. The membranes were washed several times with TBS solution and further incubated for 1 h with a 1:5,000 dilution of horseradish peroxidase-coupled, rabbit anti-goat secondary antibody for CYP2C11 and 1:5,000 dilution of donkey anti-rabbit second antibody for CYP2C23 and CYP2J2. The immunoblots were developed using an ECL detection kit (Amersham; Arlington Heights, IL). To normalize the expression of CYP isoforms, renal micosomes (10 μg) from treated and control rats were incubated with a 1:5,000 dilution of mouse anti-chicken -actin antibody (Sigma; St. Louis, MO) for 10 h. The secondary antibody was horseradish peroxidase-coupled, rabbit anti-mouse antibody (1:5,000). Immunoreactive -actin was detected as described above. The ECL films of Western blot analysis were scanned, and densitometry analysis was performed with Scion Image software using a gray color scale as a standard.

Immunohistochemistry analysis. Kidneys were isolated from virgin and pregnant rats (days 6, 12, and 19 of pregnancy, n = 5) and were cut into small slices. A specimen cup containing 2-methyl butane was precooled for 45 min in a styrofoam cooler containing dry ice and ethanol. The kidney slice was embedded in a specimen mold containing OCT compound (Miles Scientific). The specimen mold was then placed into the specimen cup for 2 min. The frozen samples were kept at –80°C until they were cut. The samples were cut in a cryostat at a thickness of 10 μm and thawed onto glass slides. Specimes were fixed in cold acetone for 10 min at –20°C. To distinguish the expression of the different CYP isoforms, CYP2C11 was detected by using diaminobenzidine (DAB), CYP2C23 was detected by red fluorescence (tetramethylrhodamine isothiocyanate, TRITC), and CYP2J was detected by green fluorescence (fluorescein isothiocyanate, FITC). For the DAB method of detection, nonspecific binding sites were blocked by 2% normal rabbit serum in PBS, and endogenous peroxidase activity was blocked by 0.9% hydrogen peroxide in PBS for one h. The slides were rinsed with PBS and incubated with a 1:100 dilution of goat anti-rat CYP2C11 antibody for 12 h at room temperature. The slides were rinsed with PBS and covered with a 1:100 dilution of a biotinylated-coupled rabbit anti-goat second antibody for 30 min. The staining of the slides was performed by an ABC kit (Vector Laboratories; Burlingame, CA), and slides were developed for 3 min using a DAB kit (Vector Laboratories). The slides were lightly counterstained with hematoxylin and examined by microscopy. For the fluorescence method of detection, the slides were rinsed with PBS three times and then incubated with rabbit anti-CYP2C23 or rabbit anti-CYP2J2 primary antibody. The slides were blocked with 3.3% normal serum and then incubated with TRITC-labeled goat anti-rabbit secondary antibody for CYP2C23 or FITC-labeled goat anti-rabbit secondary antibody for CYP2J2. The slides were mounted with Vectashield (Vector Laboratories) and examined by fluorescence microscopy. The quantification of color and fluorescent images was conducted using Metamorph software (Universal Imaging; Downingtown, PA).

Protocols to Evaluate the Effect of PPOH on EET Synthesis, CYP Enzyme Expression, and Blood Pressure
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To determine the dose effect of PPOH in vivo, 8-wk-old female rats (n = 3) were injected with PPOH at doses of 10, 20, or 40 mg/kg or vehicle control (2-hydroxypropyl--cyclodextrin) for 12 h. After treatment, rats were killed, and kidneys were removed for microsomal preparations as described above. 20-HETE and EET production were determined by the HPLC method described above.
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Pregnant rats on the 15th gestational day were injected with PPOH (20 mg﹞kg–1﹞day–1 iv) for 4 days (days 15-18 of pregnancy). Pregnant rats in the control group were injected with vehicle control. After treatment, the animals underwent surgery as described above, and MAP (n = 5) was measured. Rats were killed on day 19 of gestation, and kidneys were removed for microsomal preparation for assessing CYP2C and CYP2J expression and EET production. Fetal pups were removed from pregnant rats (n = 5), and body weight was recorded.1 i- F4 A$ G6 m1 `' F
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Statistical Analysis
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Data are expressed as means ± SD. The significance of differences between groups for EET and 20-HETE production data after PPOH treatment was evaluated with ANOVA for repeated measurements followed by a Duncan's multiple-range post hoc test. All other data were analyzed by one-way ANOVA or an unpaired two-tailed t-test. Statistical significance was set at P # P2 a' e5 [9 [& r. `) k% H+ t
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RESULTS
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Changes in MAP, Renal EET Production, and CYP2C and CYP2J Expression During Pregnancy9 b3 w; k* l$ Z6 G: T+ f

MAP in pregnant rats on day 6 (120 ± 7 mmHg) was not different from that of the control virgin rats (122 ± 2 mmHg). In contrast, blood pressure was significantly decreased on the 12th (112 ± 7 mmHg, P 4 W$ a7 I, i  x* j6 A+ p

To examine the CYP enzymes responsible for the upregulation of EET production, we conducted Western blot analysis for CYP2C11, CYP2C23, and CYP2J isoforms in renal cortical microsomes isolated from pregnant rats at different stages of pregnancy and from age-matched control virgin rats. A representative Western blot of CYP2C11, CYP2C23, and CYP2J isoforms in renal cortical microsomes is shown in Fig. 1B. Densitometry analysis normalized with -actin revealed that CYP2C11 expression on days 6, 12, and 19 was increased by 14% (1.54 ± 0.2 vs. 1.35 ± 0.3 arbitrary units, n = 3), 27% (1.72 ± 0.2 vs. 1.35 ± 0.3 arbitrary units, n = 3, P

To further examine the localization of the changes of CYP2C and CYP2J expression, we conducted immunohistochemical analysis in renal sections isolated from pregnant rats on days 6, 12, and 19 of gestation and in control virgin rats. As shown in Fig. 2 for all renal sections, CYP2C11, CYP2C23, and CYP2J proteins were expressed in the renal cortex. The most intense staining was noticed in the cortical tubules, whereas glomeruli showed very low immunoreaction for CYP2C and CYP2J proteins. Renal sections indicated a significant increase of CYP2C11, CYP2C23, and CYP2J expression in the renal cortical tubules isolated from pregnant rats on days 6, 12, and 19 of gestation. The staining intensity revealed that CYP2C11 expression was significantly increased by 11% (8.8 ± 0.2 vs. 7.9 ± 0.14 arbitrary units, n = 5, P ' W" [/ G3 `( F# J! n& E/ H
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Effect of PPOH on MAP, Fetal Pup Body Weight, Renal EET Production, and CYP2C and CYP2J Expression. L+ T) l' q# W
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Experiments were carried out in virgin female rats to determine the efficacy and selectivity of PPOH. Animals were injected intravenously with vehicle or PPOH at 10–40 mg/kg; renal cortical EET and 20-HETE production were determined 12 h later. Arachidonic acid epoxygenase activity in renal cortical microsomes was decreased by 13, 24, and 37% after treatment with 10, 20, and 40 mg/kg PPOH, respectively, relative to control vehicle-treated rats (Fig. 3). In contrast, renal -hydroxylase activity was unaffected by PPOH treatment from 10 to 40 mg/kg. We therefore used 20 mg/kg of PPOH in subsequent studies because this dose is within the middle of the dose-response curve for PPOH, and it does not have significant impact on blood pressure change in control female rats (data not shown).
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Pregnant rats on day 15 of gestation were injected with vehicle or PPOH (20 mg/kg) for 4 consecutive days (from days 15 to 18 of gestation). We used this protocol to study the effect of PPOH in late pregnancy. As shown in Fig. 4, treatment with PPOH caused a significant elevation of blood pressure, from 92 ± 2 to 113 ± 13 mmHg, and a significant reduction in fetal pup body weight. Under these conditions, PPOH treatment resulted in a selective inhibition of renal cortical epoxygenase activity without a significant effect on cortical -hydroxylase activity (Fig. 5A). Western blot analysis for CYP2C11, CYP2C23, and CYP2J isoforms in the cortical microsomes further demonstrated that PPOH treatment caused a reduction in their protein levels (Fig. 5B). Densitometry analysis normalized to -actin revealed a 30% decrease in CYP2C11 (1.1 ± 0.3 vs. 1.58 ± 0.5 arbitrary units, n = 3), 35% decrease in CYP2C23 (1.56 ± 0.3 vs. 2.39 ± 0.2 arbitrary units, n = 3, P
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DISCUSSION
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Normal pregnancy in rats is associated with a significant decrease in blood pressure (1, 14), and it has been shown that the kidneys make a major contribution to the regulation of blood pressure in pregnant rats (13). However, whether EETs are involved in the regulation of blood pressure in pregnant rats is unknown. This study demonstrates that renal arachidonic acid epoxygenase activity is increased during pregnancy. This conclusion is based on the observation that the ability of renal cortical microsomes to catalyze epoxidation of arachidonic acid is significantly increased on days 6, 12, and 19 of gestation. Western blot (Fig. 1) and immunohistochemical (Fig. 2) results show that the expression of renal CYP epoxygenase enzymes, CYP2C11, CYP2C23, and CYP2J, is also significantly elevated during pregnancy in the rat. In addition, blood pressure is decreased during pregnancy. Although the blood pressure measurements for these pregnant rats were performed under anesthesia and therefore do not ideally represent normal physiological conditions as in conscious animals, these results at least provide us with some insights into blood pressure changes during pregnancy. In addition, we observed that the highest renal cortical epoxygenase activity occurs on day 12 of gestation (Fig. 1), whereas the lowest blood pressure occurs on day 19 of gestation. The reasons for the disagreement between cortical EET production and blood pressure during pregnancy are still not clear. Evidence from the literatures has accumulated demonstrating that EETs are involved in the regulation of renal epithelial transport, vascular smooth muscle cell function, and long-term blood pressure control (7, 19). The elevation of renal EET production on days 6, 12, and 19 of gestation can decrease renal resistance and sodium reabsorption and these biological actions would decrease blood pressure. However, the relative EET production between renal vascular and tubular sites during pregnancy is still not clear. Further investigation to compare renal vascular and tubular EET production on different gestational days will be required to clear up this disagreement. Moreover, treatment with PPOH, a selective inhibitor for epoxygenases, during late pregnancy leads to an increase in blood pressure and a reduction of the size of fetal pups. Taken together, these results demonstrate that EETs may contribute significantly to the regulation of blood pressure during pregnancy.' h, `% G% d5 n  V8 Y9 [+ p3 ]

Numerous studies have shown that the CYP2C and CYP2J enzymes are the major arachidonic acid epoxygenases in the rat kidney and thereby are the primary contributors of EET synthesis. For example, Holla et al. (6) have demonstrated that recombinant CYP2C proteins can catalyze the epoxidation of arachidonic acid into EETs, and CYP2C11 has the highest activity. CYP2C23 has been shown to be the major arachidonic acid epoxygenase in the rat kidney (6). Similarly, recombinant CYP2J2 and CYP2J5 are active in the metabolism of arachidonic acid to EETs (15, 24). Interestingly, an upregulation of CYP2J proteins corresponds to an increase of urinary EET excretion in the kidneys of spontaneously hypertensive rats (25). Our present results demonstrating upregulation of CYP2C11, CYP2C23, and CYP2J proteins, along with the increasing activity of arachidonic acid epoxidation, suggest that these isoforms contribute significantly to the synthesis of EETs, and thus may play an important role in the regulation of renal function and blood pressure during pregnancy.
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It is well recognized that CYP-derived eicosanoids constitute an important role in the regulation of physiological and pathophysiological processes. These metabolites are formed endogenously in various tissues and exert potent biological effects on cellular functions. Studies of their role in normal and diseased cells and tissues are impeded by difficulty in selectively targeting their synthesis or effects, because these metabolites are generated from multiple closely related proteins of the CYP superfamilies (19). We have previously characterized several selective inhibitors for -hydroxylation or epoxidation of arachidonic acid in renal microsomes (23). Among them, PPOH was found to be very specific for the epoxidation reaction, with an IC50 value about 9 μM, whereas the IC50 for -hydroxylation is >200 μM (23). In the present study, we used PPOH to block the epoxidation pathway and found that PPOH specifically blocked EET production without significantly affecting 20-HETE production in female rats (Fig. 3) and in pregnant rats (Fig. 5). Similar results were observed by using another selective CYP epoxygenase inhibitor, N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) found in the literature (2). Moreover, PPOH is a suicide inhibitor, i.e., an inhibitor that resembles the substrate and inactivates the enzyme. Because CYP2C11, CYP2C23, and CYP2J isoforms were inactivated by PPOH as evidenced by reduced expression levels (Fig. 5), these results suggest that these proteins are very important enzymes for renal EET production. These enzymes may also play an important role in blood pressure regulation and other physiological functions during pregnancy because PPOH raised blood pressure and decreased the size of fetal pups (Fig. 4).% l: D) G  y. @. I. v
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Both EETs and DHETs are produced by renal tubular structures and renal microvessels and have actions that are relevant to the operation of renal mechanisms controlling renal function and blood pressure. EETs have been shown to inhibit sodium transport mechanisms in cultured epithelial cells and isolated tubular segments (5, 16, 20). In addition, Imig et al. (9) have demonstrated that the renal microvessels dilate in response to 11,12-EET and 14,15-EET, whereas the epoxide hydrolase product 11,12-DHET has no vasodilatory actions. This group has shown that reducing EET metabolism by the inhibition of epoxide hydrolase lowers blood pressure in angiotension II-induced hypertension (10). All of these results indicate that the upregulation of EETs in the renal tubules and microvessels may cause natriuresis and lower blood pressure during pregnancy. In the present study, we have shown that pregnancy caused the induction of renal EET synthesis and CYP epoxygenase expression (Figs. 1 and 2), which may play an important role in the regulation of renal function during pregnancy. It has yet to be determined whether EETs contribute to pregnancy-induced changes of renal function such as glomerular filtration rate and renal blood flow.0 E" |% ]5 y+ T+ w4 w9 w
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In summary, this study is the first to demonstrate that EETs and the expression of the enzymes that catalyze their formation are altered in the kidney during pregnancy. These data also demonstrate that PPOH, a selective epoxygenase inhibitor, inhibits renal production of EETs, downregulates the expression of CYP2C and CYP2J proteins in pregnant rats, and causes an elevation of blood pressure and a reduction of body weight of fetal pups. This study calls attention to the possibility that augmentation of EET synthesis in renal tissues during pregnancy impacts the regulation of blood pressure.
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GRANTS, F1 i& e# R, U

This study was supported by National Heart, Lung, and Blood Institute Grant R01 HL-70887 (to M.-H. Wang)./ }6 c: ~6 b2 N

ACKNOWLEDGMENTS5 o6 a2 s7 Z" ^

The authors thank Dr. D. C. Zeldin (National Institute of Environmental Health Science, Research Triangle Park, NC), who kindly provided rabbit anti-human CYP2J2 antibody, and Dr. J. H. Capdevila (Vanderbilt University, Nashville, TN), who provided rabbit anti-rat CYP2C23 antibody. The authors also thank Dr. Rhea-Beth Markowitz for editorial assistance and Dr. D. M. Pollock for comments on the discussion.

FOOTNOTES0 D4 R; S3 L2 \1 ]" [- y( T
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.8 r5 I/ v% j7 X* r+ O

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