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(Hypertension. 1996;27:281-286.)
© 1996 American Heart Association, Inc.

Articles

Activation of Angiotensin-Generating Systems in the Developing Rat Kidney

Presented in part at the Society for Pediatric Research meeting, May 5, 1994, and published in abstract form in Pediatr Res. 1994;35:363A.

Igor V. Yosipiv; Samir S. El-Dahr

From the Department of Pediatrics, Section of Pediatric Nephrology, Tulane University School of Medicine, New Orleans, La.

Correspondence to Samir S. El-Dahr, MD, Tulane University School of Medicine, Department of Pediatrics, SL-37, 1430 Tulane Ave, New Orleans, LA 70112.

	Abstract

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Abstract The present study was designed to determine the developmental changes in intrarenal angiotensin (Ang) peptides in the rat. Kidney Ang I and II levels were threefold and sixfold higher in newborn than adult kidneys, respectively (Ang I, 678±180 versus 243±38 fmol/g, P<.01; Ang II, 667±75 versus 103±6 fmol/g, P<.001). Intrarenal Ang II levels correlated positively with the temporal changes in renin gene expression (r=.93, P<.001). However, no correlation was found between renal Ang II content and angiotensin-converting enzyme (ACE) expression during development, which prompted us to evaluate whether renal enzymes, other than renin and ACE, contribute to Ang II formation in the developing kidney. Angiotensin peptide levels were measured in newborn and adult kidney homogenates incubated with human angiotensinogen (a poor rat renin substrate) for 30 minutes at 37°C. Inhibitors of aspartyl proteases and metalloproteases were ineffective in preventing the formation of Ang II in either newborn or adult kidneys. However, addition of the serine protease inhibitors soybean trypsin inhibitor and phenylmethylsulfonyl fluoride inhibited Ang II generation in the newborn kidneys only. In contrast, Ang I generation was not affected by inhibition of serine proteases in either newborn or adult kidneys. We conclude that Ang I and II synthesis is activated in the developing rat kidney. In addition to renin and ACE, the newborn rat kidney expresses serine protease activity that is capable of generating Ang II directly from angiotensinogen. This putative enzyme is induced in the newborn kidney and may cooperate with renin in the activation of Ang II synthesis during early development.

Key Words: renin-angiotensin system • enzymes • kidney • serine proteinases

	Introduction

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In the developing animal, postnatal maturation is accompanied by temporally regulated changes in the expression of the components of the intrarenal RAS.^{1 2 3 4} The temporal association of accelerated renal growth with elevated levels of renin and AT₁ receptor gene expression^{2 4} strongly suggests a role for Ang II in renal growth and maturation. However, it is unknown whether the developing kidney contains high levels of the peptide hormone Ang II. As a growth-promoting factor in vascular smooth muscle⁵ and fetal mesangial cells,⁶ Ang II may participate in nephrovascular development. In support of this hypothesis, pharmacological blockade of Ang II formation (ACE inhibition) or action (AT₁ receptor antagonists) suppresses kidney DNA synthesis and retards glomerular growth.^{7 8}

We have recently demonstrated that renal ACE mRNA and enzymatic activity are low after birth and induced during the preweaning period in the rat.³ We also found a reciprocal relationship between Ang II and ACE levels in the maturing rat kidney.⁹ Since renin synthesis is elevated,² the depressed ACE activity in the developing kidney raises the question of how the developing kidney can sustain the generation of high steady state levels of Ang II and suggests the presence of alternative pathways for angiotensin generation.

A number of studies have shown that several tissues, including heart, blood vessels, and adipocytes, can generate significant amounts of angiotensin peptides, independent of renin or ACE.^{10 11 12 13} In addition to renin, enzymes that release Ang I from angiotensinogen include reninlike aspartyl proteases such as cathepsin D and pepsin. Serine proteases of the kallikrein gene family (tissue kallikrein, tonin, SEV) can release Ang II directly from angiotensinogen, whereas chymotrypsin-like enzymes (chymase and cathepsin G) convert Ang I to Ang II independent of ACE.^{14 15 16} Accordingly, our aim was to determine whether enzymes in addition to renin and ACE are in part responsible for the formation of endogenous Ang II in the developing rat kidney.

	Methods

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Late-gestation pregnant rats and adult male Sprague-Dawley rats (weight, 200 to 250 g) (Charles Rivers Laboratories, Wilmington, Mass) were maintained on regular rat chow and tap water until the day of the study. After spontaneous delivery, newborn rats (at 1, 5, 15, 20, and 40 days of postnatal life) were studied. These age groups were chosen to encompass the developmental periods before and after the completion of nephrogenesis in the rat. After decapitation, the individual kidneys from 4 to 5 animals in each group were harvested, decapsulated, blotted dry, weighed, and immediately processed as described below. These studies were performed in accordance with the guidelines of the Animal Care Committee of Tulane University School of Medicine.

Measurement of Kidney Ang I and Ang II
Steady state kidney Ang I and Ang II contents were measured by an RIA with the use of rabbit anti–Ang I or II antiserum (Arnel) as previously described.¹⁷ The kidneys of each individual animal were homogenized in ice-cold 100% methanol (10% wt/vol). The supernatant was dried overnight in a vacuum centrifuge at 4°C, and the dried residue was redissolved in ice-cold phosphate buffer (50 mmol/L, pH 7.4). Angiotensin peptides were extracted by applying the reconstituted supernatant to a phenyl-bonded solid phase extraction (SPE) column (Bond-Elut, Analytichem); they were then washed sequentially with water (3 mL), hexane (1.5 mL), and chloroform (1.5 mL), followed by elution of angiotensin peptides from the SPE column with 1 mL 90% methanol in water (twice). The eluants were collected and stored at -20°C. Before the RIA, the eluants were evaporated to dryness under vacuum at 4°C and reconstituted in phosphate buffer and processed for measurement of steady state Ang II contents in day 1, 5, 15, 20, 40, and adult kidneys. Ang I contents were also measured in day 1, 5, 20, and adult kidneys. The reconstituted kidney extracts were incubated with antiserum and ¹²⁵I-radiolabeled Ang I or Ang II for 48 hours at 4°C. Bound and free angiotensin peptides were separated by dextran-coated charcoal, and the supernatants were counted by a computer-linked gamma counter for 5 minutes. Results are reported in femtomoles per gram kidney weight. The sensitivity of the Ang I and Ang II assays was <5 fmol/g. The percent specific binding for Ang I and Ang II was 25.6% and 27.5%, with nonspecific binding of 0.5% and 0.6%, respectively.

In Vitro Generation of Ang II From Newborn and Adult Kidneys
Kidneys from 5-day-old and adult rats were homogenized in ice-cold phosphate buffer (50 mmol/L, pH 7.4) containing renin and ACE inhibitors (10 mmol/L EDTA, 20 µmol/L pepstatin, 2.5 mmol/L 1,10-phenanthroline, 40 µmol/L enalaprilat). A portion of the homogenate was used to measure baseline Ang I and Ang II contents, while the remaining homogenates were used for the in vitro generation of angiotensin peptides from exogenous angiotensinogen. Homogenates were incubated with human angiotensinogen (Calbiochem, 100 nmol/L) at 37°C for 30 minutes since rat renin possesses very weak activity on human angiotensinogen.¹⁸

To further ensure that the Ang II formed from exogenous human angiotensinogen in this assay was not the result of endogenous rat renin or ACE, we performed the following experiments. (1) We measured the amount of Ang I generated in the presence and absence of renin and ACE inhibitors from newborn and adult kidney homogenates incubated with angiotensinogen for 30 minutes at 37°C. If rat renin contributes to the generation of Ang I in this assay, the amounts of Ang I generated at the end of incubation should be much higher in the samples that did not contain the inhibitor mixture. On the other hand, a lack of a significant difference in Ang I levels in the presence and absence of the inhibitors would indicate that the contribution of endogenous rat renin to Ang I generation from human angiotensinogen under the conditions of this assay is negligible. (2) We measured ACE activity in the presence of renin/ACE inhibitors after incubation with angiotensinogen. (3) We measured the amount of Ang II generated after incubation with human angiotensinogen in the presence and absence of renin and ACE inhibitor mixture. Again, a lack of a difference between the two values would suggest a nonsignificant role for endogenous ACE in the generation of Ang II. (4) To further document inhibition of ACE, we measured Ang II levels after adding exogenous Ang I (100 nmol/L) to the incubation in the presence of the renin/ACE inhibitor mixture. (5) Finally, to evaluate the purity of the human angiotensinogen substrate for a lack of contaminating angiotensin-generating enzymes, we measured the amount of Ang II generated after incubating 100 nmol/L of human angiotensinogen in phosphate buffer alone for 30 minutes at 37°C in the presence or absence of the renin/ACE inhibitors.

To assess the role of serine proteases in Ang II generation, kidney homogenates from 5-day-old and adult rats were incubated with human angiotensinogen (and renin/ACE inhibitors) in the presence of SBTI (10 µmol/L) or PMSF (3 mmol/L). All incubations were performed at pH 7.4 to inhibit the lysosomal cysteine proteases cathepsins B, H, and L, which are active only in an acid pH. After 30 minutes, the reaction was stopped by placing the tubes on ice, followed by solid-phase extraction in 90% methanol and Ang II measurement by RIA as described above.

The differences in the amounts of Ang II between the samples not incubated with angiotensinogen and those incubated with angiotensinogen were taken as an estimate of Ang II–releasing activity of intrarenal enzymes.

RNA Hybridization Analysis of RAS Gene Expression
Total kidney RNA was extracted according to Chomczynski and Sacchi.¹⁹ RNA was measured spectrophotometrically at 260 nm. The A_260/280 ratios were 1.9. RNA samples (30 µg) were resolved by gel electrophoresis in 1% agarose containing 2.2 mol/L formaldehyde. After vacuum blotting into a positively charged nylon membrane (GeneScreen Plus, NEN) and UV cross-linking, the integrity of RNA was assessed by visualization of 28S and 18S ribosomal RNA by UV shadowing of the membrane at 254 nm. Slot blots for renin were prepared by dissolving 1.25 to 5 µg of total RNA in 0.5 mL of sterile 25 mmol/L sodium phosphate buffer (pH 7.2). RNA was then applied onto the nylon membrane with the use of a Minifold II Slot-Blotter (Schleicher & Schuell). The blots were allowed to dry at room temperature.

The membranes were hybridized to rat renin,²⁰ angiotensinogen,²¹ and ACE²² cDNAs labeled with [³²P]dCTP by random priming. Specific activities of the probes were 1x10⁹ cpm/µg DNA. Prehybridization, hybridization, and posthybridization washes were performed as previously described.²³ After autoradiography, signal intensity was measured by densitometry (Ultrascan LKB). To correct for differences in RNA loading, the blots were stripped of the cDNAs and rehybridized to a ³²P-labeled GAPDH cDNA. All densitometric readings were factored for those of GAPDH.

Data Analysis
After autoradiography, the intensity of each signal on the slot blots was measured by optical density recorded on an XL Laser densitometer (LKB). For each individual kidney sample, three measurements of signal intensity at different dilutions of RNA (1.25, 2.5, and 5 µg) were obtained. The dilutions to be analyzed were chosen to ensure that comparisons were performed on the linear portion of the dose-response curve. Comparisons among the groups were performed by one-way ANOVA and Newman-Keuls or Scheffé's post hoc tests or by Student's t test. A value of P<.05 was considered statistically significant. All data are reported as mean±SEM.

	Results

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Developmental Changes in Kidney Angiotensin Peptide Contents
To measure steady state kidney angiotensin peptide levels, extreme care was taken to harvest the kidneys rapidly, followed by immediate homogenization in methanol to inactivate all potential angiotensin-forming enzymes. Steady state kidney Ang I contents were highest on the first day of postnatal life (678±180 fmol/g, n=4) and decreased threefold by adulthood (243±38 fmol/g, n=4, P<.05) (Fig 1A). Likewise, kidney Ang II contents were high immediately after birth (667±75 fmol/g, n=4) and on the fifth day of postnatal life (892±91 fmol/g, n=4) (Fig 1B). Kidney maturation was accompanied by a significant age-related decline in Ang II levels: threefold by day 15 (272±40 fmol/g, n=4) and sixfold by adulthood (103±6 fmol/g, n=4).

View larger version (14K): [in this window] [in a new window]   Figure 1. Bar graph shows immunoreactive Ang I (A) and Ang II (B) levels in the kidneys of Sprague-Dawley rats during postnatal development. D indicates day. *P<.001 vs other age groups.

Developmental Changes in RAS Gene Expression
The postnatal expression of angiotensinogen, renin, and ACE genes in the kidney is shown in Fig 2 (n=4 to 5 animals per group). Angiotensinogen mRNA was barely detectable on day 1 of postnatal life and underwent biphasic changes thereafter: a peak was observed on day 5, followed by a marked reduction on days 15 and 20 and a second peak in the adult (day 80). By Northern analysis, renin mRNA levels were elevated on days 1 and 5 and decreased thereafter. Renin mRNA levels were 10-fold higher in the newborn than adult kidneys, whereas ACE mRNA levels were low after birth and increased postnatally, peaking on days 15 to 20 (Fig 2). By slot blot analysis, expression of ACE mRNA increased threefold from day 5 to day 15 of postnatal life (10±1.5 versus 33±6 densitometric units; P<.05), confirming our previous results.³ The latter study also showed that ACE activity was low on days 1 and 5 (0.1 to 0.2 nmol/mg protein per minute) and increased fivefold by day 15 (1.0 nmol/mg protein per minute).³ As we previously reported,³ GAPDH expression did not change significantly during postnatal kidney development (Fig 2).

View larger version (31K): [in this window] [in a new window]   Figure 2. Developmental expression of RAS genes in the kidney. Northern blots of total kidney RNA (30 µg per sample) pooled from 4 to 5 animals per group were hybridized to radiolabeled rat angiotensinogen, renin, and ACE cDNAs and washed under very stringent conditions. The ACE autoradiogram was overexposed to permit the appearance of the signal on day 1, which masked the visual difference in the signals between days 5 and 15. Slot blot analysis showed a threefold increase in ACE mRNA from day 1 to day 15 consistent with our previous results (data not shown; Reference 3). Hybridization to a GAPDH probe served as a control for RNA loading. D indicates day; Ad, adult.

A positive correlation was found between kidney renin mRNA and Ang II contents during postnatal kidney development (r=.93, P<.001) (Fig 3). No significant correlations were observed between kidney ACE mRNA and Ang II contents.

View larger version (15K): [in this window] [in a new window]   Figure 3. Line graph shows temporal correlation between renin mRNA levels (from slot blot analysis) and Ang II contents in the rat kidney. A positive correlation was observed (r=.93, P<.001).

In Vitro Generation of Ang I and Ang II
To estimate the renin/ACE-independent Ang II–forming activity, we compared baseline kidney Ang II contents with in vitro generated Ang II values after incubation in homogenates obtained from the same animals with exogenous human angiotensinogen. Human angiotensinogen was used as the exogenous substrate because endogenous rat renin has very low activity on human angiotensinogen.¹⁸ Accordingly, most of the generated Ang II can be presumed to have resulted from cleavage of human angiotensinogen by enzymes other than rat renin. The kidneys were homogenized in a phosphate buffer to maintain the enzymatic activity of all potential angiotensin-forming pathways. Baseline Ang I levels in phosphate buffer–homogenized kidneys were significantly higher in the newborn than adult rats (31.5±3.9 versus 5.0±0.3 pmol/g, P<.001, n=4). Similarly, baseline Ang II levels were higher in the newborn kidney (1.5±0.1 versus 0.5±0.03 pmol/g, P<.001, n=4) (Table 1).

View this table: [in this window] [in a new window]   Table 1. Angiotensin Peptide Generation In Vitro From Human Angiotensinogen Incubated With Newborn and Adult Rat Kidney Homogenates

To demonstrate that endogenous rat renin has no significant role in angiotensin peptide generation from the human angiotensinogen substrate, newborn and adult kidney homogenates were incubated for 30 minutes at 37°C with human angiotensinogen (100 µmol/L) in the absence of the renin/ACE inhibitor mixture (n=4 per group) (Table 1). At the end of incubation without angiotensinogen, Ang I levels increased 10-fold in the newborn and 7.5-fold in the adult. The inclusion of angiotensinogen in the incubation mixture did not result in any significant changes in the amount of Ang I generated compared with incubation without angiotensinogen in either newborns or adults. Furthermore, the increase in Ang I levels was not affected by the addition of renin or ACE inhibitors. These findings indicate that newborn and adult rat kidneys do not possess the Ang I–generating enzymes specific for human angiotensinogen. In contrast, as shown in Table 1, newborn and adult kidneys are capable of generating Ang II from human angiotensinogen. Ang II generation from human angiotensinogen was not suppressed by the renin/ACE inhibitors.

The efficacy of ACE blockade by the inhibitor cocktail in the in vitro incubation system was evaluated. As shown in Table 2, ACE activity in the homogenates, determined from the hydrolysis of hippuryl-histidyl-leucine, was inhibited by more than 80% in the newborn and by 50% in the adult (n=4 per group) groups. Furthermore, the addition of exogenous Ang I to the homogenates (in the presence of the inhibitor mixture) did not result in any significant increase in Ang II generation, indicating efficient blockade of ACE activity (n=4 per group) (Table 2).

View this table: [in this window] [in a new window]   Table 2. ACE Activity and Conversion of Ang I to Ang II in the In Vitro Incubation System in the Presence or Absence of Renin/ACE Inhibitor Mixture

We evaluated whether the human angiotensinogen substrate contains any contaminating Ang II–generating activity. For this purpose, angiotensinogen (100 nmol/L) was incubated in phosphate buffer (pH 7.4) for 30 minutes at 37°C either alone or in the presence of the renin/ACE inhibitor cocktail and Ang II was assayed at the end of incubation (n=3). Only small amounts of Ang II were detected (without inhibitors, 87±16 fmol/mL; with inhibitors, 152±2 fmol/mL). Thus, the human angiotensinogen substrate did not contain any significant Ang II–generating activity.

Developmental Changes in Renal Ang II–Forming Activity
As shown in Fig 4, incubation of kidney homogenates with human angiotensinogen resulted in a marked increase in Ang II levels compared with baseline values. Coincubation with the serine protease inhibitors SBTI or PMSF prevented the generation of Ang II in the newborn kidneys. In contrast, Ang I levels did not change after incubation with human angiotensinogen (9.4±2.8 versus 9.7±1.5 pmol/g), nor were they affected by the serine protease inhibitors (9.3±2.7 pmol/g). These findings, together with those in Table 2, strongly suggest that serine protease activity generates Ang II directly from angiotensinogen. It must be pointed out here that the marked increases in Ang II generation after incubation with angiotensinogen and the inhibition by serine protease inhibitors were observed in all experiments (n=4). However, the relative increases in Ang II generation varied among the experiments up to 10-fold.

View larger version (12K): [in this window] [in a new window]   Figure 4. Bar graph shows effect of SBTI and PMSF on the generation of Ang II by 5-day-old kidneys homogenized in phosphate buffer (50 mmol/L, pH 7.4) and incubated with angiotensinogen (Ao) (100 nmol/L). *P<.01 vs other groups. The inhibition of Ang II generation by SBTI and PMSF indicates that the putative Ang II–generating enzyme is a serine protease.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that the rat kidney RAS genes have unique patterns of developmental expression. Whereas renin gene expression is activated in the newborn kidney, ACE gene expression is low after birth and rises during the preweaning period. Angiotensinogen gene expression during postnatal life in the rat is biphasic. This study also documents that angiotensin peptide synthesis is activated in the developing kidney and clearly shows that the developmental changes in kidney Ang I and II correlate strongly with the temporal changes in renin gene expression, indicating that renin plays a critical role in determining the activity of the intrarenal RAS during postnatal development. However, the elevated levels of Ang II peptide in the developing kidneys, in the face of low ACE expression and activity,³ suggest that other enzymes may also be involved in the generation of Ang II in the developing kidney.

In the present study we tested the hypothesis that alternate pathways for Ang II formation are present in the rat kidney and are activated during early development. Although angiotensinogen is the only substrate for renin, angiotensinogen can be cleaved to Ang I by enzymes other than renin. These enzymes include cathepsin D, pepsin, and other aspartyl proteases and reninlike enzymes.^{10 11} Furthermore, ACE-independent conversion of Ang I to Ang II can be demonstrated in several tissues and species, including the rat hindlimb,²⁴ hamster cheek pouch,²⁵ dog and monkey pulmonary and mesenteric arterial strips,²⁶ and human heart and detrusor muscle.^{27 28 29} ACE-independent Ang II–forming activity in dog blood vessels and human heart is blocked by inhibitors of serine proteases.^{30 31 32} On the contrary, Ang I to Ang II conversion in rat blood vessels is totally dependent on ACE.³³ There is also growing evidence that Ang II can be released directly from angiotensinogen by enzymes that include tonin,^{14 15 16 34} related serine proteases, tissue kallikrein,^{14 16 35} SEV,^{14 36 37 38} and possibly other members of the kallikrein gene family.

Using this in vitro system, we found that the newborn and adult kidneys possess a significant Ang II–generating system that was not inhibited by inhibitors of aspartyl and metalloproteases. In contrast, the Ang II–forming activity was inhibited completely by SBTI and PMSF in the newborn but not in adult kidneys, indicating that the enzyme mediating Ang II formation in the newborn kidney is a serine protease. The nature of the nonrenin, non-ACE, Ang II–forming enzyme in the adult kidney remains to be elucidated. Unlike Ang II, Ang I levels did not change after incubation with angiotensinogen, suggesting that increased Ang II formation via this pathway originates directly from the substrate angiotensinogen.

To examine the role of enzymes other than renin or ACE in Ang II formation in the kidney, we measured the in vitro generation of Ang II from human angiotensinogen (a poor rat renin substrate) incubated with newborn and adult kidney homogenates. Additional evidence demonstrating that Ang II generation from human angiotensinogen incubated with rat kidney homogenates is renin and ACE independent include the following: (1) Ang I generation was not different in the presence or absence of human angiotensinogen in the incubation mixture, and it was not inhibited by aspartyl protease inhibitors; (2) ACE inhibitors significantly reduced ACE enzymatic activity and prevented the formation of Ang II from exogenous Ang I; and (3) human angiotensinogen substrate does not contain angiotensin-forming enzymes.

The present study was not designed to elucidate the identity of the Ang II–forming serine proteases in the developing kidney. However, candidate enzymes include tonin, SEV, and tissue kallikrein. Using in vitro translation of tissue polyA⁺-enriched RNA followed by immunoprecipitation with a tonin antibody and sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, Woodley-Miller et al³⁹ identified a toninlike immunoreactivity in the adult rat kidney, suggesting that tonin may be expressed in the kidney. There is also evidence that SEV and tissue kallikrein mRNAs are expressed in the newborn rat kidney,^{36 40} although the ontogenic changes in kallikrein (low in the fetus and newborn and increasing with development) do not fit the developmental pattern of Ang II generation in the newborn.

In summary, the present study demonstrates that Ang II–generating systems (both renin dependent and renin independent) are activated in the developing rat kidney. In addition to renin and ACE, the rat kidney possesses serine protease activity that appears to form Ang II directly from angiotensinogen and is induced during early development. This alternative pathway of Ang II formation may be important in mediating the activation of the RAS in conditions associated with low ACE activity such as during the early newborn period.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme Ang I, Ang II = angiotensin I, angiotensin II PMSF = phenylmethylsulfonyl fluoride RAS = renin-angiotensin system RIA = radioimmunoassay SBTI = soybean trypsin inhibitor SEV = submandibular enzymatic vasoconstrictor

	Acknowledgments

 
This study was supported by a National Kidney Foundation young investigator grant (Dr El-Dahr) and by a Research Fellowship Grant from the American Heart Association, Louisiana Affiliate (Dr Yosipiv). We are grateful to Dr L. Gabriel Navar's laboratory, in particular Annie Hymel, for teaching us the angiotensin peptide RIA, to Dr William Baricos for helpful discussions during the course of this study, and to Drs Guillermo Scicli and Luis Carbini (Henry Ford Hospital, Detroit, Mich) for critical reading of the manuscript.

Received March 8, 1995; first decision April 24, 1995; accepted November 4, 1995.

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