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(Hypertension. 1995;25:1129-1134.)
© 1995 American Heart Association, Inc.

Articles

Potassium Negatively Regulates Angiotensin II Type 1 Receptor Expression in Human Adrenocortical H295R Cells

Ian M. Bird; R. Ann Word; Colin Clyne; J. Ian Mason; William E. Rainey

From the Departments of Obstetrics and Gynecology, Biochemistry, and Physiology and The Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, and the Departments of Obstetrics and Gynecology, Meriter Hospital, University of Wisconsin, Madison (I.M.B.).

	Abstract

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Abstract We have previously shown that the human adrenocortical H295R cell line expresses the type 1 angiotensin II receptor (AT₁-R) and that expression of this receptor is downregulated at the level of mRNA by forskolin or dibutyryl-cAMP as well as by angiotensin II (Ang II). In this study we examine the effects of K⁺ on both AT₁-R mRNA and receptors, as monitored through ¹²⁵I–Ang II binding in the presence of PD 123319. After treatment with a maximal stimulatory steroidogenic dose of K⁺ (14 mmol/L), H295R cells showed an increase in cytosolic free Ca²⁺ from 113 to 212 nmol/L. Unlike the effects of Ang II, this increase could be abolished by pretreatment with the Ca²⁺ channel antagonist nifedipine (1 µmol/L). AT₁-R mRNA levels also fell in response to elevated extracellular K⁺ in a dose-dependent (K_d, 9 mmol/L; maximal fall in message at 12 mmol/L) and time-dependent (maximum 50% at 12 hours) manner. The change in AT₁-R mRNA level was less rapid than that in response to activation of phosphoinositidase C by Ang II or adenylyl cyclase by forskolin or by dibutyryl-cAMP. Unlike the action of Ang II but similar to the action of forskolin or dibutyryl-cAMP, the action of K⁺ was sustained. Changes in mRNA level in response to treatment with K⁺, Ang II, or dibutyryl-cAMP were also paralleled by changes in ¹²⁵I–Ang II binding in each case. The mechanism of action of K⁺ on AT₁-R mRNA also appears to be mediated through the opening of voltage-sensitive channels on the plasma membrane because the drop in AT₁-R mRNA was similarly abolished by the Ca²⁺ channel blocker nifedipine. In conclusion, our findings show that AT₁-R mRNA levels can be controlled through a Ca²⁺-dependent signaling pathway, as well as through phosphoinositidase C or adenylyl cyclase signaling pathways, and that these changes in mRNA level underlie a corresponding change in receptor protein at the cell surface.

Key Words: angiotensin II • receptor • potassium • adrenal • human • calcium

	Introduction

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The renin–angiotensin II (Ang II) system is a central component of the hormonal mechanisms regulating blood pressure as well as fluid and electrolyte homeostasis. At least two subpopulations of Ang II receptors (designated AT₁ and AT₂) have been identified as a result of the use of nonpeptide Ang II antagonists, and it is now clear that the AT₁ receptor (AT₁-R), coupled to phosphoinositidase C, is primarily involved in the control of vasoconstriction and electrolyte homeostasis.¹ The recent cloning of the AT₁-R from several species has made it possible to begin to define the mechanisms underlying hormonal regulation of AT₁-R expression.^{2 3} Because of obvious limitations in the availability of tissues, few studies have been concerned with hormonal regulation of AT₁-R expression in humans. However, studies in primary cultures of human adrenocortical cells have shown that AT₁-R mRNA level is reduced by Ang II treatment.⁴ A human adrenocortical tumor cell line (H295R) is also available that expresses AT₁-R functionally coupled to phosphoinositidase C and secretes aldosterone in response to Ang II.⁵ The level of AT₁-R mRNA is also reduced in these cells after treatment with activators of both adenylyl cyclase and phosphoinositidase C,⁶ in agreement with the findings in human adrenocortical cell primary cultures.⁴ These changes in AT₁-R mRNA are also analogous to that seen for rat AT_1a-R mRNA in rat kidney mesangial cells⁷ but different from AT_1b mRNA in the rat adrenal.^{8 9 10} However, while putative AT_1a and AT_1b mRNA species have also been isolated in humans, AT_1b mRNA was not detectable in human adrenal.¹¹ Thus the finding that AT_1b mRNA and AT₁-R levels in the rat adrenal increase rather than decrease in response to Ang II infusion, low sodium diet, or to a lesser extent high potassium diet^{8 9 10} probably relates to the expression of different gene products in the adrenals of each species.

We have recently reported that H295R cells secrete aldosterone in response to elevated K⁺,¹² which is known to regulate aldosterone secretion in vivo independently of adenylyl cyclase or phosphoinositidase C activation.¹³ In the present study we demonstrate further that elevated K⁺ acts to increase cellular Ca²⁺ through opening voltage-sensitive channels and that increased cellular Ca²⁺ alone is also sufficient to promote a dose-dependent and sustained decrease in AT₁-R mRNA as well as the AT₁-R itself in human adrenocortical H295R cells.

	Methods

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Unless otherwise stated, laboratory reagents were obtained from Sigma Chemical Co. The agonists and antagonists used were KCl, [Asp¹,Ile⁵]-Ang II, nifedipine, forskolin, dibutyryl-cAMP (dbcAMP), and the selective AT₂ receptor antagonist PD 123319 (Parke Davis). Fura 2-AM was obtained from Molecular Probes Inc.

Cell Culture
H295R cells were initially obtained as NCI-H295 cells from the American Type Culture Collection (ATCC) and then selected as described previously.¹² Because of growth and culture differences between the original ATCC cells and the selected subpopulation, these cells are designated as H295R cells. The H295R cells are available from ATCC as catalog No. CRL 2128. Cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F-12 media (DME/F-12 containing pyridoxine HCl, L-glutamine, and 15 mmol/L HEPES; Gibco BRL, catalog No. 11331-014) supplemented with insulin (6.25 µg/mL), transferrin (6.25 µg/mL), selenium (6.25 ng/mL), linoleic acid (5.35 µg/mL; 1% ITS plus, Collaborative Research), 2% low-protein serum replacement-1, and antibiotics. Cells were maintained and grown on 75-cm² flasks (Costar) at 37°C under an atmosphere of 5% CO₂/95% air. Cells were subcultured, and after 48 hours medium was removed and replaced with serum-free medium (DME/F-12 containing antibiotics and 0.01% bovine serum albumin [BSA]). Cells were cultured for a further 24 hours before treatment in the same medium.

Determination of [Ca²⁺]_i in Fura 2–Loaded H295R Cells
Cells were plated onto glass coverslips and cultured in growth medium as described for 3 days. Cells were then loaded with fura 2-AM ester (5 µmol/L, Molecular Probes) in buffer (mmol/L: NaCl 130, KCl 4.8, MgCl₂ 1, CaCl₂ 1.5, Na₂HPO₄ 1, glucose 15, and HEPES 10, as well as 1 mg/mL BSA, pH 7.4). Loading was achieved over 45 minutes at 37°C under a 5% CO₂/95% O₂ atmosphere. Cells were then rinsed and incubated for a further 20 minutes in the same buffer. Coverslips were mounted on a polytetrafluoroethylene frame in a cuvette containing buffer without fura 2 or albumin. The frame ensured a 30° angle of the coverslip to the incident/excitation fluorometer beam. All fluorescence measurements were made with a Perkin-Elmer 650-10S fluorescence spectrophotometer while the buffer was stirred with an electronically controlled paddle to ensure rapid mixing of added reagents. Excitation and emission wavelengths were 340 nm (slit width, 5 nm) and 510 nm (slit width, 5 nm), respectively. Autofluorescence of cells and buffers was measured and subtracted when necessary. Cytosolic free calcium ([Ca²⁺]_i) was computed with the formula [Ca²⁺]_i=K_d(F-F_min)/(F_max-F), where K_d=224 nmol/L; F is observed 340-nm fluorescence; F_max is 340-nm fluorescence at a [Ca²⁺] (millimoles per liter) sufficient to saturate fura 2; and F_min is 340-nm fluorescence at a [Ca²⁺] (nanomoles per liter) sufficient to give no significant binding to fura 2.^{14 15} A single excitation wavelength was used because the fluorometer was not equipped with an automated filter wheel. Concentrations calculated by this method did not differ by more than 5% from those calculated by the ratio method when excitation wavelength was changed manually.¹⁶

Northern Analysis for AT₁-R mRNA Levels
Cells on 100-mm culture dishes were lysed into 1 mL RNAzol B solution (Cinna Biotecx) and transferred to a microfuge tube. Phase separation was achieved by mixing with 0.15 mL CHCl₃, incubation at 4°C for 5 minutes, and centrifugation (12 000g, 20 minutes, 4°C). The upper phase (0.7 mL) was transferred to a second microfuge tube, and RNA was then precipitated by the addition of 0.8 mL isopropanol and standing for 1 hour at -20°C. RNA was recovered by centrifugation (30 minutes, 12 000g, 4°C), and the recovered pellet was washed once in 75% ethanol (1.0 mL) before being dried under air and dissolved in 1 mmol/L EDTA, pH 7.0 (0.1 mL). After recovery and purity were determined by measurement of absorbance at 260 and 280 nm, samples were precipitated by the addition of 1 mL absolute ethanol and 0.01 mL sodium acetate (3 mol/L, pH 5.2) and stored at -70°C until analysis.

Samples were subjected to electrophoresis on gels containing 1.1% agarose (Bio-Rad) in the presence of formaldehyde. The presence and integrity of the major RNA species were examined under UV light to ensure consistency between lanes. RNA was transferred to a Magna NT membrane (MSI) by pressure blotting (75 psi, 1 hour; PossiBlot Pressure Blotter, Stratagene) and cross-linked under UV light. Prehybridization was carried out at 42°C overnight in a final buffer composition of 50% formamide, 5x SSC (20x SSC contains 3.0 mol/L NaCl and 0.3 mol/L trisodium citrate, pH 7.0), 1x PE (5x PE contains 250 mmol/L Tris-HCl, pH 7.5, 0.5% sodium pyrophosphate, 5% sodium dodecyl sulfate [SDS], 1% polyvinylpyrrolidone, 1% Ficoll, 25 mmol/L EDTA, and 1% BSA), and 50 µg/mL tRNA. Hybridizations were performed in the same buffer at 42°C for 16 to 24 hours with bovine AT₁-R antisense probe labeled by asymmetrical polymerase chain reaction with [³²P]dCTP (Amersham).⁶ The blots were then washed in 2x SSC containing 0.1% SDS at room temperature for 15 minutes and in 0.1x SSC containing 0.1% SDS at room temperature for 2x30 minutes before being dried and quantified by direct radioimaging (AMBIS Systems, 12-hour direct exposure time) and then exposed to film (Hyperfilm, Amersham). Blots were subsequently stripped and reprobed for GAPDH mRNA. An antisense probe was prepared by asymmetrical polymerase chain reaction amplification of the human cDNA (bases 43-478) in the presence of [³²P]dCTP, and hybridization and posthybridization wash conditions were exactly as described above. Specific hybridization was then evaluated by direct radioimaging as above. Results for AT₁-R mRNA levels were corrected for minor variations in sample loading (according to the GAPDH content of each lane) before being expressed as percent AT₁-R mRNA in the control sample of each experiment (100%).

Radiolabeled Ang II Binding Studies
Ang II receptor binding was determined on cultured cells as described previously.⁶ After treatment of cells on 12-well plates for the times indicated, cells were rinsed free of any bound agonists by brief incubation in acidified medium (pH 4.0, 5 minutes, 4°C). Cells were then rinsed once more in neutral medium before incubation with radiolabeled Ang II (¹²⁵I, 2000 Ci/mmol, Amersham, 100 000 cpm per well), together with 5x10^-10 mol/L unlabeled Ang II, and 1 µmol/L PD 123319 in 0.3 mL binding medium (DME/F-12 containing 0.5% BSA and 0.1% bacitracin, pH 7.4) for 1 hour at 37°C. At the end of this time, wells were washed in DME/F-12 medium (4°C, three times) before cell lysis in 0.5 mol/L NaOH containing deoxycholate (0.4%). Receptor-bound radioactivity associated with the cell lysates was then determined in a gamma counter.

Statistical Analysis
Statistical analysis was accomplished with ANOVA followed by Student-Newman-Keuls multiple comparison analysis.

	Results

Top Abstract Introduction Methods Results Discussion References

 
To confirm that the mechanism of action of elevated K⁺ on the H295R cell was indeed mediated through the opening of plasma membrane voltage-dependent channels, we studied the effect of K⁺ on [Ca²⁺]_i in cells preloaded with fura 2 (Fig 1). Treatment with KCl (14 mmol/L) resulted in an increase in [Ca²⁺]_i from 113±1.5 to 212±19.3 nmol/L (n=6). The initial rise in [Ca²⁺]_i was less than that observed in response to Ang II, rising from 123.5±1.4 to 331±33.7 nmol/L (n=8). Cells were also pretreated with nifedipine (1 µmol/L) for 3 minutes, and responses to Ang II or K⁺ treatment were recorded. Nifedipine pretreatment abolished the response to K⁺, suggesting that the effects of K⁺ were mediated through a voltage-sensitive Ca²⁺ channel in the plasma membrane. The initial increase in [Ca²⁺]_i (first 30 seconds) in response to Ang II was only partially attenuated by nifedipine (from 331±33 to 175±4 nmol/L), and the later sustained increase in [Ca²⁺]_i was abolished (Fig 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Traces show effects of K+ and angiotensin II (AII) on [Ca2+]i in H295R cells. Cells preloaded with fura 2 were stimulated with K+ (14 mmol/L) or angiotensin II (10 nmol/L), and changes in [Ca2+]i were monitored at 510 nm as described in "Methods." Stimulation of cells was performed with agonist alone (A) or after pretreatment with nifedipine (1 µmol/L for 3 minutes, B). Additions were made at the times indicated by arrows. Results are representative of data obtained from three independent experiments.

Northern analysis of RNA from H295R cells treated for 20 hours with elevated K⁺ showed a dose-dependent reduction in mRNA level for the AT₁-R (Fig 2), with half-maximal reduction in mRNA levels at 9 mmol/L K⁺. At this time, the maximum fall in message level was to 50% of that seen in controls, which exceeded the reduction after treatment for 20 hours with Ang II but not dbcAMP. The time dependency of the response to K⁺, Ang II, dbcAMP, and forskolin is shown in Fig 3. Elevated K⁺ promoted a fall in AT₁-R mRNA level that was maximal by 12 hours, representing a slower response than that seen after Ang II, dbcAMP, or forskolin treatment. The AT₁-R mRNA level remained low during prolonged treatment, similar to the level observed after treatment with dbcAMP or forskolin but not treatment with Ang II, where the message level returned to 80% of control by 24 hours.

View larger version (26K): [in this window] [in a new window]   Figure 2. Plots shows dose dependency of decrease in angiotensin II type 1 receptor (AT1-R) mRNA level in response to elevated extracellular K+ in H295R cells. Cells were treated with elevated K+ at the doses shown or with dibutyryl-cAMP (dbcAMP, 1 mmol/L) for 20 hours in serum-free medium before extraction of total RNA and Northern analysis (25 µg RNA per lane) as described in "Methods." After hybridization with the AT1-R probe (top panel), the membrane was stripped and reprobed for GAPDH (G3PDH in figure) (bottom panel) to verify loading consistency. Graphic results are from three independent experiments (mean±SEM, normalization to the level of GAPDH as described in "Methods"); *P<.05 relative to control. Autoradiographic results show representative data obtained in one of these experiments. AII indicates angiotensin II.

View larger version (18K): [in this window] [in a new window]   Figure 3. Line graphs show time dependency of decrease in angiotensin II type 1 receptor (AT1-R) mRNA level in response to elevated extracellular K+ (14 mmol/L), angiotensin II (AII, 10 nmol/L), dibutyryl-cAMP (dbcAMP, 1 mmol/L), or forskolin (10 µmol/L) in H295R cells. Cells were treated for indicated times in serum-free medium before extraction of total RNA and Northern analysis (25 µg RNA per lane). After hybridization with the AT1-R probe, the membrane was stripped and reprobed for GAPDH to verify loading consistency (±10%). Results are normalized to the GAPDH level detected in each lane and expressed as mean±SEM of data from three independent experiments for each treatment. *P<.05 relative to control.

Having observed K⁺ downregulation of AT₁-R mRNA levels in H295R cells, we investigated whether this was also paralleled by changes in Ang II receptors on the cell surface. Our ¹²⁵I–Ang II binding assay was performed in the presence of PD 123319, making the assay specific to changes in the AT₁-R only. Time-dependent changes in AT₁-R binding sites in response to each treatment (Fig 4) were reminiscent of the changes in mRNA (Fig 3). Treatment with K⁺ resulted in a gradual but otherwise sustained decline in receptor binding, reaching a minimum level of 60% of control by 24 hours. Forskolin also promoted a sustained but otherwise greater loss in receptor binding (45% of control by 12 hours), but the effects of both K⁺ and forskolin contrasted to the effect of Ang II, which promoted an initial decline in receptor binding, reaching a minimum of 40% of control by 12 hours, and then returned to control levels by 24 hours.

View larger version (12K): [in this window] [in a new window]   Figure 4. Line graphs show time dependency of decrease in angiotensin II type 1 receptor (AT1-R) level in response to elevated extracellular K+ (14 mmol/L), angiotensin II (AII, 10 nmol/L), or forskolin (10 µmol/L) in H295R cells. Cells were treated for indicated times in serum-free medium before the AT1-R–specific 125I–Ang II binding assay was performed as described. Results are mean±SEM of data from three (K+, AII) or four (forskolin) independent experiments for each treatment. *P<.05.

To confirm that K⁺ treatment reduced AT₁-R mRNA levels by promoting the influx of extracellular Ca²⁺, we investigated the effect of nifedipine on this response. The reduction of AT₁-R mRNA at both 4 and 20 hours in response to K⁺ could be fully reversed by nifedipine at 1 µmol/L (Fig 5). However, nifedipine failed to completely reverse the action of dbcAMP on AT₁-R mRNA (4 or 20 hours) or the effects of Ang II (4 hours). Furthermore, the Ca²⁺ channel agonist Bay K8644 was able to reduce AT₁-R mRNA in a dose-dependent manner, and at a maximally effective dose (1 µmol/L) AT₁-R mRNA was decreased to a level comparable to that in response to K⁺ (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 5. Graphs show effect of nifedipine on K+-stimulated fall in angiotensin II type 1 receptor (AT1-R) mRNA levels in H295R cells. Cells were treated for 4 hours (left) or 20 hours (right) in serum-free medium in the absence (control) or presence of K+ (14 mmol/L) and nifedipine (Nif, 1 µmol/L or as indicated). Cells treated with angiotensin II (AII, 10 nmol/L) or dibutyryl-cAMP (dbcAMP, 1 mmol/L) were also included as positive controls as indicated. Total RNA extraction and Northern analysis were then performed as described in "Methods" (see also Fig 2). Results show data from two of four separate experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Since the initial cloning of the AT₁-R cDNA, attention has focused on the possibility that the expression of the receptor protein may be regulated at least in part at the level of the cellular mRNA. Studies on the hormonal regulation of AT₁-R binding and AT₁-R mRNA levels in the human H295R adrenocortical cell line⁶ and human adrenocortical cell primary cultures⁴ have shown that a decrease in message is observed in response to both Ang II and factors that activate adenylyl cyclase or otherwise promote an increase in cAMP. These findings for H295R cells⁶ are similar to those reported in rat kidney mesangial cells (which primarily express rat AT_1a mRNA)⁷ but differ from those in rat adrenal, where AT_1b mRNA levels increase in response to Ang II infusion, low sodium diet, or to a lesser degree high potassium diet.^{8 9 10} However, while putative AT_1a and AT_1b mRNA species have also been isolated in humans, AT_1b mRNA was not detectable in human adrenal,¹¹ and the corresponding gene has yet to be isolated.

In the adrenal gland, aldosterone formation is not only regulated by Ang II and corticotropin but also by increased circulating K⁺. The mechanism of action of increased K⁺ appears to be through the opening of voltage-sensitive Ca²⁺ channels and thus stimulates short-term aldosterone production by a mechanism distinct from that of Ang II and corticotropin.¹³ The H295R cell line also exhibits an increase in aldosterone secretion in response to elevation of extracellular K⁺¹² as well as Ang II⁵ or activation of adenylyl cyclase.¹² We have previously shown that the effect of Ang II on AT₁-R mRNA levels was fully reproduced at 4 hours by a combination of calcium ionophore (A23187) and phorbol ester but nevertheless could be partially reproduced by calcium ionophore alone.⁶ We therefore considered the possibility that elevation of extracellular K⁺, a physiological agent known to regulate calcium channel activity in adrenal cells, could also lead to alteration of AT₁-R or its corresponding mRNA levels in the human adrenal cortex.

In the present study we have shown that elevation of extracellular K⁺ results in an increase in [Ca²⁺]_i in H295R cells that was completely dependent on the opening of dihydropyridine-sensitive Ca²⁺ channels. This differed from the response to Ang II, which initially involved the more rapid release of an intracellular pool of Ca²⁺, followed by influx of extracellular Ca²⁺ through nifedipine-sensitive voltage-gated channels. In addition we also found that elevation of extracellular K⁺ resulted in a reduction of AT₁-R mRNA levels that was both concentration dependent (half-maximal response, 9 mmol/L; maximal, 12 mmol/L) and time dependent, and the drop in AT₁-R mRNA was sustained below 50% of control on prolonged treatment. This contrasts with the recovery in AT₁-R message level observed on prolonged treatment with Ang II but was similar to that observed for treatment with either dbcAMP or forskolin. The mechanism of action of K⁺ on AT₁-R mRNA appears to be mediated entirely through the Ca²⁺ signaling pathway, in that the effect of K⁺ on AT₁-R mRNA was abolished by the calcium channel blocker nifedipine and could be reproduced by the calcium channel agonist Bay K8644. The action of K⁺ through a calcium-dependent signaling pathway alone may also explain the slower time course for the initial fall in AT₁-R mRNA when compared with that seen after Ang II treatment, which would also activate the protein kinase C signaling pathway.

The time-dependent changes in AT₁-R in response to K⁺, Ang II, and forskolin treatment, measured with a specific binding assay, confirm that the cell surface receptor apparently downregulates with only a few hours of delay behind the changes in mRNA in each case. This finding confirms that elevated K⁺ can alter receptor expression at the cell surface as well as at the level of mRNA and also extends our previous finding to show that whereas Ang II has little long-term effect on binding, the transient reduction in AT₁-R mRNA in response to Ang II treatment is followed by a similar transient reduction in AT₁-R. Thus the level of cell surface receptor appears to correlate directly with the level of AT₁-R mRNA, regardless of the cell signaling pathway involved. This at least is consistent between species because in the rat adrenal an increase in AT_1b mRNA in response to low sodium or high potassium diet¹⁰ is also paralleled by a corresponding increase in AT₁-R, as seen by both Western analysis¹⁰ and receptor binding studies.^{17 18}

These results, together with previously reported findings,⁶ lead us to conclude that in H295R adrenocortical cells, both AT₁-R and its corresponding mRNA levels are controlled through three distinct signaling pathways, namely, protein kinase A, protein kinase C, and a Ca²⁺-dependent signaling pathway. These findings may indicate why in many mammalian species the AT₁-R density is reduced in the zona fasciculata/reticularis relative to the zona glomerulosa.^{19 20 21} The finding that long-term treatment with elevated K⁺ as well as agents that activate adenylyl cyclase brings about a sustained reduction in AT₁-R expression further suggests that physiological agents that can stimulate aldosterone secretion independently of the renin-angiotensin system may be able to selectively desensitize the adrenal zona glomerulosa to Ang II in humans. Furthermore, since Ang II action on adrenocortical cells is also known to attenuate expression of 17-hydroxylase,^{22 23 24 25} the consequence of AT₁-R downregulation may be an increase in 17-hydroxylase expression and so further development of zona fasciculata function, with corresponding cortisol secretory capacity.

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (DK 43140 to W.E.R.), the American Heart Association, Texas Affiliate (93R-082 to W.E.R.), and Merck Sharp & Dohme. I.M.B. was supported in part by National Institutes of Health Training Grant No. T32-HD-07190.

	Footnotes

 
Reprint requests to Dr W.E. Rainey, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9032.

Received October 26, 1994; first decision November 16, 1994; accepted March 8, 1995.

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