(Hypertension. 1997;29:98.)
© 1997 American Heart Association, Inc.
Research Articles (Issue 1, Part 1) |
Vascular Angiotensin-Converting Enzyme Expression Regulates Local Angiotensin II
the Franz Volhard Clinic Virchow Klinikum, and the Max Delbrück Center for Molecular Medicine, Humboldt University of Berlin (Germany); University of Erlangen-Nürnberg (Germany); and Institut National de la Santé et de la Recherche Médicale (INSERM) U367, Paris, France.
Correspondence to Friedrich C. Luft, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, FRG.
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Abstract |
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We tested the hypothesis that changes in angiotensin-converting enzyme (ACE) gene expression can regulate the rate of local vascular angiotensin II (Ang II) production. We perfused isolated rat hindlimbs with an artificial medium and infused renin and Ang I via the perfusate. Ang I and II were measured by radioimmunoassay. We then increased ACE gene expression and ACE levels in the rat aorta by producing two-kidney, one clip (2K1C) hypertension for 4 weeks. Gene expression was measured by RNAse protection assay, and ACE activity in the vessel wall was measured by the Cushman-Cheung assay. Angiotensin I infusion at 1, 10, 100, and 1000 pmol/mL led to 371±14 (±SEM), 3611±202, 44 828±1425, and 431 503±16 439 fmol/mL Ang II released, respectively, from the hindlimbs (r=.98, P<.001). Thus, the conversion rate did not change across four orders of magnitude, and the system was not saturable under these conditions. In 2K1C hindlimbs, Ang I infusion (0.5 pmol/mL) resulted in increased Ang II generation (157±16 versus 123±23 fmol/mL, P=.014 at minute 10) compared with controls. ACE gene expression and ACE activity were increased in 2K1C hindlimbs compared with controls (36±4 versus 17±1 mU/mg protein, P<.001). Ang II degradation in the two groups did not differ. To investigate the conversion of locally generated Ang I, we infused porcine renin (0.5 milliunits per mL) into 2K1C and control hindlimbs. Despite markedly higher Ang I release in sham-operated than in 2K1C rats (71±8 versus 37±6 pmol/mL, P=.008 at minute 12), Ang II was only moderately increased (36±3 versus 25±6 pmol/mL, P=.12 at minute 12). This difference between 2K1C rats and controls reflected a higher rate of conversion in 2K1C rats. Thus, Ang I conversion in the rat hindlimb is linear over a wide range of substrate concentrations and occurs at a fixed relationship. Nevertheless, increased ACE gene expression and ACE activity in the vessel wall lead to an increase in the conversion of Ang I to Ang II. We conclude that local ACE gene expression and ACE activity can influence the local rate of Ang II production.
Key Words: angiotensin-converting enzyme • renin • angiotensin • hypertension, renovascular • hindlimb
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Introduction |
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Angiotensin-converting enzyme (ACE) is the major enzyme responsible for generating the vasoconstrictor peptide angiotensin II (Ang II) from Ang I. Vascular ACE is a membrane-bound enzyme1 2 3 localized on endothelial cells4 and under certain circumstances in the smooth muscle cells and adventitial layers of blood vessels.5 6 Numerous studies have shown that the vessel wall is an important site of vasoactive peptide generation and metabolism.2 3 7 8 9 10 11 12 13 Interest in ACE as a regulator of Ang II production has been intensified by genetic studies linking the ACE gene locus to hypertension in rats and to several cardiovascular diseases in humans. In humans, an insertion/deletion (I/D) ACE gene polymorphism has been linked to an increased risk for myocardial infarction,14 cardiomyopathy,15 and left ventricular hypertrophy16 but not for hypertension.17 18 The I/D polymorphism is associated with increased plasma and cellular ACE levels.19 20 However, the mechanisms linking the ACE gene to cardiovascular disease remain unclear. One plausible hypothesis is that the plasma and/or tissue ACE levels influence Ang II concentration in the peripheral and/or local circulation. On the other hand, the Km of the ACE–Ang I reaction is several orders of magnitude higher than even extremely high Ang I levels. ACE–Ang I in vitro kinetics suggest that at normal or elevated Ang I levels, the rate of Ang II generation should depend only on the substrate concentrations, as suggested by kinetic studies with artificial ACE substrates in isolated perfused lungs.21 However, in the Heidelberg stroke-prone spontaneously hypertensive rat, in which the ACE gene locus has been linked to hypertension and vascular ACE gene expression is increased, neither vascular ACE activity nor vascular Ang II generation from Ang I was altered.22 23 24 Thus, the mechanism by which increased ACE should lead to increased Ang II formation is not clear. We selected two different approaches to test the hypothesis that vascular ACE regulates Ang II generation. We first attempted to saturate the ability of vascular ACE to generate Ang II by infusing increasing Ang I doses into Sprague-Dawley rat hindlimbs and measuring the Ang II generated. We then upregulated vascular ACE by inducing two-kidney, one clip (2K1C) hypertension and used the hindlimbs to study the conversion of Ang I to Ang II.
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Methods |
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Animals
Male Sprague-Dawley rats (Ivanovas, Kislegg, FRG) weighing 300 to 400 g were used for all experiments. The rats were kept in rooms at 24±2°C, fed a standard rat diet (No. C-1000, Altromin) containing 0.2% sodium by weight, and allowed free access to tap water. All procedures were done according to guidelines of the American Physiological Society and were approved by local authorities (permits AZ IV A4/5-G 0399/92 and AZ IV A4/5-G 0398/92).
2K1C Renal Hypertension Initiation
For induction of 2K1C renovascular hypertension, rats (180 to 190 g) were anesthetized with chloral hydrate (0.4 g/kg IP, Sigma Chemical Co). The left renal artery was occluded by a silver clip (0.2 mm ID); the right kidney was not disturbed. Sham control rats were sham-operated by an incision made in the flank after a 30-second interruption of renal artery blood flow. Four weeks after clipping, systolic pressure was measured by tail-cuff plethysmography with rats under light ether anesthesia.
Hindquarter Perfusion
Preparation and perfusion were performed with rats under thiopental anesthesia (75 mg/kg IP, Trapanal, BYK-Gulden) as previously described.7 13 Briefly, rats underwent median laparotomy. After evisceration, the abdominal aorta and inferior vena cava were cannulated, and perfusion was begun immediately. A more cranial part of the aorta was excised, rinsed in cold NaCl solution, frozen in liquid nitrogen, and stored at -80°C. The hindquarters were perfused in a nonrecirculating system with a modified Tyrode's solution containing 2 g/L glucose and 20 g/L of the artificial colloid Ficoll 70 (Pharmacia). The perfusate was gassed with a mixture of 95% O2/5% CO2, adjusted to pH 7.4, and maintained at 37°C. Hindquarter perfusion was performed at a constant flow rate of 10 mL/min with a peristaltic pump (Abimed Gilson), and perfusion pressure was monitored by a pressure transducer connected to an on-line computer system (TSE). Protocols were started after 30 minutes of equilibration perfusion. All experimental substances were infused into the perfusion system at a rate of 100 or 200 µL/min by means of a syringe pump (HT Infusors).
Tissue Preparation and Measurement of ACE Activity
Aortic segments were homogenized at 4°C in 1 mL cold phosphate-buffered saline (120 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L phosphate buffer, pH 7.4) containing 8 mmol/L of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) (Sigma) with the use of a potter homogenizer (Braun). The crude homogenate was centrifuged at 600g for 10 minutes at 4°C. Five microliters of the supernatant was used for assay. ACE activity was determined by hydrolysis of the synthetic substrate p-benzoyl-L-glycyl-L-histidyl-L-leucine (Hip-His-Leu, Bachem). The assay conditions were 5 mmol/L substrate, 100 mmol/L potassium phosphate (pH 8.3), and 300 mmol/L NaCl in a total volume of 250 µL with incubation at 37°C, as described by Cushman and Cheung.25 The reaction was stopped by addition of 50 µL of 12% (wt/vol) phosphoric acid. The amount of hippuric acid liberated from the substrate was analyzed by high-performance liquid chromatography.20 One unit of activity was defined as the amount of enzyme catalyzing the release of 1 µmol hippuric acid per minute. Total protein concentration was measured by the method of Bradford26 with bovine serum albumin (Sigma) as the standard and the use of Coomassie brilliant blue G-250 (Bio-Rad).
Experimental Protocols
After an initial 30-minute period of baseline perfusion, perfusate (1 mL) for peptide measurement was collected in tubes containing 50 µL of a mixture of phenanthroline (26 mmol/L) and EDTA (125 mmol/L) (both from Sigma), taken at 5-minute intervals, beginning 5 minutes before the Ang I or porcine renin infusion.
Ang I Conversion and Ang II Formation in Rats
We investigated the formation of Ang II from infused Ang I (Sigma) over a range of concentrations in rat hindquarters. Each rat hindquarter (n=5) and a second identical perfusion channel without a hindquarter were perfused simultaneously according to the same protocol. Cumulative doses of Ang I (1, 10, 100, 1000 pmol/mL) were infused for 10 minutes followed immediately by the next higher dose. Perfusate was also collected from blank channels at the same times and served to quantify Ang I metabolism.
Ang I Conversion and Ang II Formation in Rats With Induced ACE Activity
The vascular conversion of infused Ang I to Ang II was measured in a model with increased vascular ACE gene expression, the 2K1C rat. Hindquarters of 2K1C (n=5) and matched sham-operated (n=8) rats were prepared simultaneously and mostly perfused in parallel. Ang I (0.5 pmol/mL) was infused for 30 minutes, and Ang II release was determined over 30 minutes. In another set of experiments, we investigated the degradation and uptake of Ang II in 2K1C and sham rats (n=5 each). In the same model, we also investigated the conversion of Ang I that had been generated locally within the vascular tissue from endogenous angiotensinogen (n=5 each). For induction of local Ang I formation, 0.5 milliunits per mL porcine renin (Sigma) was infused for 12 minutes; collection of angiotensin peptides was continued up to 40 minutes. Captopril (10-5 mol/L) (Sigma) was added from minute 20 to minute 40. All samples were immediately frozen on dry ice and stored at -80°C until assayed.
Enzyme Kinetic Determinations and Radioimmunoassay
Immunoreactive Ang I and Ang II concentrations in the perfusate were determined by direct radioimmunoassay.27 The cross-reactivity of anti–Ang I to Ang II was less than 0.01% and was less than 0.01% to [Val5]Ang II, 100% to [des-Asp1]Ang I, 0.03% to Ang I/II-(1-7), and 0.02% to Ang III. Values for anti–Ang II were 6% for Ang I and 70% for [Val5]Ang II, 0.1% for [des-Asp1]Ang I, less than 0.01% for Ang I/II-(1-7), and 70% for Ang III. Peptides (Bachem) represent human species. Both assays can detect 2 to 3 fmol angiotensin in 100 µL perfusate. Mean intra-assay variabilities were 9% for Ang I and 11% for Ang II, and mean interassay variabilities were 13% and 15%, respectively. Captopril did not interfere in these immunoassays.
Tissue Preparation and Ribonuclease Protection Assay
Total RNA was isolated from rat aortas by the lithium chloride precipitation technique.28 Detection and quantification of mRNA specific for ACE was performed by a ribonuclease protection assay with an Ambion RPA III kit (ITC Biotechnology GmbH) according to the manufacturer's protocol. To prepare antisense RNA probe complementary to rat ACE mRNA, we used a 375-nucleotide Pst I cDNA fragment specific for the ACE cDNA fragment subcloned into a pBluescript II KS+ vector and linearized for T3 RNA polymerase transcription by BamHI restriction enzyme.29 The probe was labeled with [32P]UTP to a specific activity of 6.2x108 cpm/µg RNA and spanned 446 nucleotides, including vector-encoded sequences. We controlled for intra-assay variability by cohybridization of the aortic RNA probes with a [32P]UTP-labeled antisense RNA probe specific for rat ß-actin, an uninfluenced housekeeping gene. For this purpose, a 150-nucleotide rat ß-actin cDNA fragment subcloned into a pBluescript II SK+ vector30 was linearized by Xba I and transcribed by T7 RNA polymerase at a specific activity of 3.2x108 cpm/µg RNA. Forty-four nucleotides of noncoding vector sequences were added during transcription. Twenty micrograms of total aortic RNA was hybridized together with 4.2x104 cpm of ACE-specific and 4.3x104 cpm of ß-actin–specific antisense probe overnight at 42°C. After combined RNAse A/T1 digestion, the protected fragments were separated by electrophoresis on a 5% denaturing polyacrylamide gel and quantitatively analyzed by densitometry with a FUJIX BAS 2000 PhosphoImager system after 24 hours of autoradiography. The signal was calculated as the ratio of ACE mRNA to ß-actin mRNA.
Data Analysis
Ang I conversion is expressed as the ratio of molar concentration of Ang II and molar concentration of Ang I measured in the hindquarter effluent. Data are expressed as mean±SEM. The relation between exogenously infused Ang I and released Ang II was assessed with simple regression analysis. Repeated measures ANOVA was used to study the influence of arterial renal clipping and time of perfusion on angiotensin release and conversion rate. Statistical significance was estimated between groups by one-way ANOVA and Scheffé's test. The numerical probability (P) values, indicating the probability of the null hypothesis, are given in the results.
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Results |
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The perfusion pressure of isolated Sprague-Dawley rat hindquarters ranged from 28 to 30 mm Hg. Ang I and Ang II were not detected in samples taken before Ang I infusion. Increasing concentrations of Ang I led to a dose-dependent release of Ang II from the perfused hindquarters (Fig 1) (n=5, correlation between extracted Ang I and released Ang II, r=.98, P<.001), which was reduced by 95% 5 minutes after infusion of captopril (10-5 mol/L). No Ang II was detected in the respective blank channels during Ang I infusion. Ang I extraction did not change with the different Ang I concentrations during infusion. The conversion rate, expressed as the ratio of Ang II to Ang I, was constant during infusion of 1, 10, and 100 pmol/mL Ang I (average ratio, 2.15).
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Systolic pressure was significantly elevated in renovascular hypertensive rats (198±5 mm Hg, n=15) compared with sham-operated controls (121±2, n=29; P<.001). The body weight of 2K1C rats (313±12 g, n=15) was slightly lower than that of age-matched sham-operated rats (337±7, n=29; P=.057). Aortic rat angiotensinogen and ACE gene expression were determined by mRNA protection assay. Rat angiotensinogen mRNA levels did not change within the groups (2K1C, n=5; sham, n=6; P=.28). The ACE mRNA signal in aortas of 2K1C rats was higher than in controls (2K1C, n=5; sham, n=6; P=.08) (Fig 2). Aortic ACE activity was much higher in 2K1C rats (n=5) than in sham-operated controls (n=12, P<.001) (Fig 3).
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During infusion of 0.5 pmol/mL Ang I, more Ang II was released from 2K1C (n=5) rats than from controls (n=8, P=.02 at minute 20) (Fig 4). In contrast, the degradation and uptake of infused Ang II into hindquarter preparations was unchanged (n=5 each, P=.51 at minute 10) (Fig 5). These data show that the difference in Ang II production between 2K1C and control hindlimbs cannot be attributed to differences in Ang II degradation. Porcine renin (0.5 microunits per milliliter) induced local Ang I and II formation in 2K1C and sham-operated control rats (Fig 6). After the renin infusion was stopped, Ang I decreased to undetectable levels. Ang I levels were higher in sham-operated rats than in the clipped group (n=5 each, P=.02 at minute 6), as would be expected, because the high renin in the early phase of 2K1C hypertensive rats depletes their endogenous angiotensinogen. In contrast, Ang II formation continued after the renin infusion was stopped up to the start of captopril administration and then decreased to undetectable levels. Despite the markedly higher Ang I levels in sham-operated than 2K1C rats, Ang II was only moderately increased (Fig 6, top and middle). This difference reflects a higher conversion of Ang I to Ang II in 2K1C rats, as expressed as the ratio of Ang II to Ang I (Fig 6, bottom). This conversion rate decreased during the experiment (n=5 each, P=.006 at minute 6, P=.044 at minute 9, and P=.38 at minute 12) (Fig 6). The ratios of Ang I conversion were quite different after the local generation of Ang I induced by renin compared with exogenous Ang I infusion (average ratios, 0.53 for renin versus 2.15 for Ang I).
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Discussion |
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The important findings of this study were that infusion of cumulative doses of Ang I into the hindlimb circulation resulted in a linear increase in Ang II generation over a wide range of concentrations. Nevertheless, upregulation of the vascular ACE gene with 2K1C hypertension demonstrated that increased ACE expression effected a higher conversion rate of exogenous Ang I to Ang II. Furthermore, when we induced local Ang I formation by infusing porcine renin, which is able to cleave rat angiotensinogen, we again observed a higher conversion rate of Ang I to Ang II in 2K1C rats.
The conversion of Ang I to Ang II in situ in the vasculature, measured with the natural substrate Ang I, apparently depended only on the Ang I levels available. This finding would support the notion that ACE is not a rate-limiting step for Ang II generation. However, despite these considerations, our experiments in 2K1C rat hindquarters demonstrated that increased ACE expression effected a higher conversion rate of exogenous and locally generated Ang I to Ang II. Therefore, our data provide direct evidence that increased vascular ACE does indeed lead to increased local Ang II formation.
The interest in ACE and its local activity has increased markedly with the discovery of the I/D human ACE gene polymorphism. The D allele is associated with higher tissue and plasma ACE levels19 20 31 in several cardiovascular diseases.14 15 16 32 The same could not be shown for polymorphisms in the renin gene,33 34 even though renin is considered to be a rate-limiting step in Ang II production and ACE is generally not. We reasoned that ACE must play some rate-limiting role in Ang II production, provided that the higher ACE levels associated with the D allele exert their effect via Ang II generation. We understand that in humans Ang I is converted to Ang II by other enzymes in addition to ACE, such as chymase, particularly in the human heart. Okunishi et al35 have drawn attention to the marked species difference in the vascular Ang II–forming pathways when humans and rodents are compared. Thus, direct comparisons between these species must be made with caution.
The physiological regulation of ACE occurs at a considerably slower rate than that of renin. In vitro or in vivo changes of endothelial ACE activity are observed only after prolonged exposure (several hours to days) to a stimulus.36 37 38 Although ACE activity is regulated slowly, the enzyme is nevertheless a determinant of Ang II production. The kinetics of the ACE–Ang I interaction are rather slow. Indeed, plasma Ang I concentration is 2- to 10-fold higher than that for plasma Ang II under normal circumstances. Moreover, variations in ACE concentration in local systemic vascular beds could regulate the local rate of tissue Ang II production, which may subsequently regulate local tissue function and structure, including vascular or cardiac remodeling.
Certain studies have addressed the question of whether chronic increases in tissue ACE expression and activity affect Ang II–mediated effects.5 36 38 Okamura et al38 performed contraction studies in isolated aortas and mesenteric arteries in the chronic phase of renovascular hypertension and showed an increased response to Ang I. They concluded that an increased local vascular Ang I conversion was the most likely explanation. Arnal et al5 demonstrated an increased ACE activity in rat thoracic aorta. Our data confirm and extend these findings. We directly measured the generation of the vasoactive effector Ang II after infusion of exogenous Ang I and after renin infusion in an intact and complete vascular bed to determine the Ang I conversion rate. We found a higher Ang II generation in 2K1C rats after administration of exogenous Ang I and a higher rate of conversion when we induced local Ang I formation by renin infusion. The ratio of Ang II to Ang I was quite different depending on whether Ang I was infused exogenously or formed locally in response to renin infusion. We speculate that this difference may represent a different conversion of locally formed Ang I, as discussed previously.7 Thus, upregulation of ACE gene expression increased the conversion rate of both infused and locally formed Ang I.
The peripheral vasculature is known to be an important site of Ang I conversion in several species,7 8 9 10 11 12 13 including humans.8 39 Since the extraction rates for Ang I across vascular beds are high, it is likely that local vascular ACE, rather than pulmonary ACE, is the main source of Ang II reaching the peripheral arterioles.7 8 10 40 41 42 We relied on Ang I infusion to the hindlimb perfusion model for several reasons. First, the natural rat substrate has a higher affinity for the enzyme than artificial substrates.1 Second, ACE acts as a membrane-bound enzyme in vivo1 as well as in our in vitro preparation. Third, we measured the conversion of Ang I across an intact and complete vascular bed instead of restricting the observation to a single vessel. Finally, we avoided the standardization procedures that pose a problem for measurements in homogenates. We believe that our approach to measurement of vascular ACE in situ most closely resembles the physiological action of the enzyme in vivo. This notion is also supported by the close similarity of vascular angiotensin conversion in our model and in the vessels of human volunteers.8
The possible contribution of non–ACE-dependent enzymatic pathways6 35 43 44 45 to the conversion rate across the hindquarter vascular bed might be a disadvantage of our approach. However, the extent to which these non–ACE-dependent pathways contribute to Ang II generation or secretion remains to be established. Furthermore, our experiments with captopril in normal rats did not provide evidence for a noteworthy contribution of non-ACE pathways to Ang II formation. A second limitation of our model is the fact that the apparent conversion of Ang I, as calculated from Ang II levels in the venous effluent, is influenced by the degradation and uptake of newly formed Ang II in the vasculature. In our study, the increased conversion of Ang I to Ang II in 2K1C rats was not due to changes in Ang II degradation and uptake, which remained unchanged. Thus, the increased Ang II release must reflect increased Ang II formation.
We were initially surprised that we could not saturate ACE and that the relationship between Ang I and Ang II perfusate concentrations remained linear. We next increased the expression of the ACE gene and its product. We relied on 2K1C hypertension, which has been documented to increase ACE gene expression within the aorta and kidney.6 38 46 We were able to show a marked increase in aortic ACE activity in 2K1C rats compared with controls. We relied on an RNAse protection assay to monitor ACE gene expression in 2K1C rats and found that ACE mRNA was slightly higher (n=5, P=.08) than in control rats. The numbers of rats we examined in these technically tedious assays were relatively small. We believe that it is highly likely that the increase in vascular ACE activity we observed was a function of increased ACE gene expression rather than translation, although we cannot rule out the latter possibility.
Not only ACE but also tissue angiotensinogen levels may determine vascular Ang II production. Since the early phase of renovascular hypertension is characterized by high plasma renin activity and decreased plasma angiotensinogen levels,46 the uptake of angiotensinogen into the vascular wall may in turn be diminished. We were unable to measure angiotensinogen release from our hindlimb preparation by our enzyme kinetic assay because the levels were below the detection limit. However, we assume that vascular angiotensinogen levels were also lower, because the infusion of porcine renin resulted in considerably lower Ang I levels in 2K1C than in control rats. Nevertheless, despite higher Ang I levels in sham-operated rats, Ang II was only moderately higher than in 2K1C rats. The conversion rate, expressed as the ratio of Ang II to Ang I, was significantly increased in 2K1C rats. However, the relationship of Ang II to Ang I decreased toward the end of the porcine renin infusion in our study. This observation may be due to substrate consumption in 2K1C rats because of lower levels of angiotensinogen and a higher rate of Ang I conversion. Thus, the rate of Ang I conversion in 2K1C rats was increased after both Ang I and porcine renin infusion.
Using the same perfusion model as in the present study, we13 23 and others47 have previously investigated Ang I conversion in many pathophysiological situations. 2K1C hypertension is the first model to show a clear-cut increase in vascular Ang I conversion. Our data challenge the conclusion of Kato et al47 that plasma renin is the sole determinant of vascular Ang II formation. ACE clearly contributes to the regulation of vascular Ang II formation in 2K1C rats. Morishita et al48 demonstrated that transfection of ACE cDNA into vascular smooth muscle cells resulted in an increased ACE activity and a parallel Ang II–mediated cellular hypertrophy. The cotransfection of renin and ACE cDNAs led to a synergistic effect. The group suggested that both renin and ACE are rate limiting in Ang II generation and function. Rakugi et al37 also reported that ACE expression was locally induced after balloon angioplasty injury experiments in rat carotid artery or abdominal aorta. The level of vascular ACE correlated with the size of the neointima.
In summary, our data show that the conversion of Ang I to Ang II in a resistance vessel bed, measured in situ by perfusion with the natural substrate Ang I, remains linear even at very high substrate concentrations. This observation, however, does not mitigate against a regulatory role of ACE for local Ang II generation, as demonstrated by our experiments in 2K1C rat hindlimbs. Increased vascular ACE expression and its activity led to a higher conversion rate of Ang I to Ang II for both infused and locally formed Ang I. Further studies will be necessary to investigate the influence of variants of the ACE gene on vascular ACE expression and Ang II formation.
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Acknowledgments |
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This study was supported by a grant-in-aid from the Bundesministerium für Bildung und Forschung. Karl F. Hilgers is the recipient of a Deutsche Forschungsgemeinschaft research fellowship (Hi 510/5-1). This work completes in part the requirements for the PhD degree of Dominik N. Müller. We gratefully acknowledge the expert technical assistance of Regina Uhlmann, Irene Strauss, Gabriele Born, Anita Müller, and Christel Lipka.
Received April 1, 1996;
first decision May 28, 1996; first decision July 8, 1996;
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