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-hydroxylase activity
with ABT reduces blood pressure in the SHR
1 Departments of
Biopharmaceutical Sciences and Pharmaceutical Chemistry, The
mechanism-based cytochrome P-450 (CYP) inhibitor
1-aminobenzotriazole (ABT) was characterized as an inhibitor of renal arachidonic acid metabolism and administered to spontaneously hypertensive rats (SHRs) to determine the effect of reduced eicosanoid production on mean arterial pressure (MAP). A single intraperitoneal dose of ABT to Sprague-Dawley rats caused a dose-dependent loss of
renal CYP content, arachidonic acid metabolism, and CYP4A protein. In
the cortex and outer medulla, ABT showed a high degree of selectivity for the CYP4A enzymes, reflected by the potent inhibition of 19- and
20-hydroxyeicosatetraenoic acid (19- and 20-HETE) formation. A 50 mg/kg
dose of ABT reduced cortical 20-HETE formation to 16.1 ± 0.82% of
control and outer medullary 20-HETE formation to 23.8 ± 0.45% of
control. In contrast, there was no inhibition of renal epoxygenase activity at this dose. Renal CYP content, arachidonic acid
cytochrome P-450 4A; 20-hydroxyeicosatetraenoic acid; renal eicosanoids
INCREASINGLY RECOGNIZED as autocrine and paracrine
mediators of renal function and vascular tone are cytochrome
P-450 (CYP)-derived eicosanoids. Major products
of renal CYP metabolism of arachidonic acid include the The effects of CYP eicosanoids on renal function and vascular tone
suggest that these metabolites play an important role in the regulation
of blood pressure. However, the contrasting effects of these
eicosanoids on renal vascular tone and tubular ion secretion make it
difficult to predict their role in vivo in regulating blood pressure.
The vasoconstrictive effects of 20-HETE on the vasculature would be
expected to be prohypertensive, whereas the inhibition of renal tubular
Na+ reabsorption would be
antihypertensive. In the spontaneously hypertensive rat (SHR), renal
arachidonic acid Limited information is available about the in vivo role of the CYP
eicosanoids in the SHR. Administration of the heme oxygenase inducers
stannous chloride and heme arginate to 7-wk-old SHRs significantly
inhibited CYP-mediated arachidonic acid metabolism in renal microsomes
and reduced the blood pressure to levels found in age-matched WKY rats
(16, 31). In one case the decrease in CYP metabolism was shown to be
specific for 1-Aminobenzotriazole (ABT) is a mechanism-based CYP inhibitor.
Inactivation of CYP enzymes by ABT requires catalytic formation of
benzyne, which then alkylates the prosthetic heme group (27). ABT has a
broad substrate specificity and in vivo inactivates 70-80% of the
total hepatic CYP content of phenobarbital-induced rats (27). ABT also
inactivates CYP protein in pulmonary and renal microsomes (17, 20).
Inhibition of the hepatic CYP4A enzymes by ABT has been characterized
both in vitro and in vivo using lauric acid as a substrate.
Preincubation of rat hepatic microsomes or treatment of rat hepatocytes
with ABT results in a time- and concentration-dependent inhibition of
lauric acid The ability to examine the in vivo significance of CYP catalyzed
arachidonic acid metabolism in blood pressure regulation in the SHR has
been hindered by the lack of inhibitors with in vivo activity. We
report here the identification of ABT as an inhibitor of renal CYP
arachidonic acid metabolism. ABT shows selectivity in the kidney for
the Materials.
Intramedic polyethylene tubing (PE-10) and Tygon tubing were purchased
from Clay Adams (Parsippany, NJ). Ketamine HCl was from Aveco (Fort
Dodge, IA), and acepromazine maleate and xylazine-20 were from Butler
(Kansas City, MO and Columbus, OH). ABT and 1-hydroxybenzotriazole were
obtained from Sigma Chemical (St. Louis, MO). Radiolabeled arachidonic
acid was purchased from Amersham Life Science (Arlington Heights, IL)
or from NEN Life Science Products (Boston, MA). HPLC solvents and
ScintiVerse LC were from Fisher Scientific (Pittsburgh, PA). All other reagents were of the highest grade
available and were purchased from Fisher Scientific or Sigma Chemical.
Animal surgery and treatment.
Male SHRs weighing 110-150 g at 6 wk of age (Charles River
Laboratories, Wilmington, MA) or male Sprague-Dawley rats weighing 170-200 g (Simonsen Laboratories, Gilroy, CA) were maintained under controlled housing conditions of light (6 AM-6 PM) and
temperature (22°C) and received standard laboratory chow and water
ad libitum. Rats were allowed at least 3 days to become acclimated to
the housing conditions before use in experiments, and blood pressure and arachidonic acid metabolism were measured at 7 wk of age. All
animal protocols were approved by the University of California San
Francisco Committee on Animal Research and followed the National Institutes of Health Guide for the Care and Use of
Experimental Animals.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
- and (-1)-hydroxylase activity, and CYP4A protein levels gradually return to control levels by 72 h after a single dose of ABT.
Cortical 20-HETE formation recovered from 17.9 ± 3.15% of control
at 6 h to 84.8 ± 4.67% of control at 72 h after ABT administration. A single injection of ABT to 7-wk-old SHRs caused an
acute reduction in MAP, which remained suppressed for at least 12 h.
The effect was maximal within 4 h and averaged 17-23 mmHg during
the 4- to 12-h period after administration. 20-HETE formation was
inhibited 85% in the cortex and 70-80% in the outer medulla during the period when MAP was reduced. A structurally related ABT
analog 1-hydroxybenzotriazole had no effect on blood pressure or renal
arachidonic acid metabolism. These results identify ABT as a selective
inhibitor of renal CYP4A activity and provide further support for a
role for 20-HETE in the regulation of blood pressure.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
- and
(-1)-hydroxylated metabolites, 20- and 19-hydroxyeicosatetraenoic
acid (20- and 19-HETE), respectively, and regio- and stereoisomeric
epoxyeicosatrienoic acids (EETs) (15). 20-HETE is a major metabolite in
renal microsomal preparations from rat, rabbit, and human and has been
implicated in a number of functions related to vascular tone and ion
transport. Canine renal arteries and proximal and distal portions of
rat afferent arterioles are constricted by 20-HETE in a dose-dependent
manner (10, 14). This vasoconstrictive effect is mediated, at least in
part, by inhibition of the opening of a large-conductance, calcium-activated potassium channel, which leads to depolarization of
the arteriole vascular smooth muscle (36). Proximal tubular Na+-K+-ATPase
and the
Na+-K+-2Cl
cotransporter in the medullary thick ascending limb of the kidney are
both inhibited by 20-HETE, resulting in natriuresis and diuresis (1,
23). 20-HETE has also been implicated in the autoregulation of renal
blood flow and tubuloglomerular feedback (37, 38). The effects of
19-HETE on renal vascular tone and ion transport contrast with those of
20-HETE, as 19(S)-HETE stimulates Na+-K+-ATPase
and 19(R)-HETE is a modest vasodilator (6, 14). Epoxide metabolites of
arachidonic acid are produced in similar quantities as 20-HETE in rat
renal microsomal preparations and are also implicated in the regulation
of ion transport and vascular tone (15). Both 5,6- and 11,12-EET as
well as 11,12-dihydroxyeicosatrienoic acid inhibit
Na+-K+-ATPase
activity in rat proximal convoluted tubules, presumably leading to
natriuresis (23). In a rat juxtamedullary nephron preparation
superfusion with 11,12- or 14,15-EET caused dose-dependent vasodilation, whereas 5,6-EET caused vasoconstriction (9). EET-induced
vasodilation is associated with an increased open-state probability of
a calcium-activated potassium channel and hyperpolarization of the
vascular smooth muscle (3). These effects have led to the proposal that
EETs are endothelial-derived hyperpolarizing factors, although this may
be species and tissue specific.
-hydroxylation is increased relative to age-matched
normotensive Wistar-Kyoto (WKY) rats (8, 13, 22). Increased 20-HETE
formation in SHR renal microsomes is maximal in young animals (8, 13, 22) and is accompanied by a decreased diameter of interlobular arteries
and afferent arterioles (8). These differences in arachidonic acid
metabolism between WKY and SHR renal microsomes appear to be specific
because 19-HETE formation shows similar changes as 20-HETE, whereas no
changes in epoxygenase activity have been reported (22). Members of the
CYP4A enzyme family are responsible for arachidonic acid - and
(-1)-hydroxylation and are expressed at high levels in the rat
kidney (15). Recently, we showed that increased expression of the
CYP4A3 and CYP4A8 genes and CYP4A immunoreactive proteins account for
the increased arachidonic acid - and (-1)-hydroxylation in the
young SHR kidney, suggesting that altered CYP4A expression in the
prehypertensive SHR kidney may contribute to the changes in renal
function during this period (13).
-and (-1)-hydroxylation, thus implicating these
pathways in blood pressure control (16). However, the interpretation of
these studies is limited because heme oxygenase inducers also affect
the production of carbon monoxide and may interact with nitric oxide
synthase, distinct systems that are known to modulate vascular tone.
20-HETE has also been implicated in the regulation of blood pressure in
the Dahl salt-sensitive model of hypertension, although its proposed
mechanism is distinct from that in the SHR. A decreased formation of
20-HETE in outer medullary microsomes of prehypertensive Dahl
salt-sensitive rats relative to the salt-resistant controls is
associated with an elevated loop chloride transport in these
animals (35).
- and (-1)-hydroxylation (11, 28). A single
intraperitoneal injection of ABT to Sprague-Dawley rats inhibits lauric
acid - and (-1)-hydroxylation more than 70% and requires at
least 5 days for complete recovery of this activity (26). Similar
information regarding the inhibition of renal CYP4A enzymes by ABT is
not available. ABT is particularly useful to probe the physiological roles of CYP enzymes because it is water soluble and is relatively nontoxic on chronic administration (18).
- and (-1)-hydroxylation of arachidonic acid and causes a
loss of CYP4A apoprotein. Inhibition of renal 20-HETE formation by ABT
is associated with a decrease in mean arterial pressure (MAP),
providing evidence for the involvement of this eicosanoid in the
regulation of blood pressure.
METHODS
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Abstract
Introduction
Methods
Results
Discussion
References
80°C. Urine and plasma samples were stored at
20°C.
Clinical chemistry analysis. Urinary Na+ and K+ levels were determined by standard flame photometry techniques, and urinary and plasma creatinine levels were determined colorimetrically by the clinical laboratory of the General Clinical Research Center at the University of California San Francisco. Creatinine clearance was calculated from the ratio of the 24-h urinary excretion rate of creatinine to the plasma concentration of creatinine at the midpoint of the urine collection period.
Preparation of microsomes and CYP determination. Microsomes were prepared from liver, renal cortex, or outer medulla samples from a single animal as described previously (13). Microsomal protein concentrations were measured by the Pierce BCA protein assay (Pierce Chemical, Rockford, IL) with BSA as the standard. Total CYP content in hepatic or renal cortical microsomes was measured from the reduced carbon monoxide difference spectra on an Aminco DW-2000 UV-VIS spectrophotometer as described by Omura and Sato (24).
Microsomal arachidonic acid metabolism.
Renal cortical and hepatic arachidonic acid metabolism was measured in
incubations containing
[1-14C]arachidonic
acid (10 µM, 0.2 µCi), microsomal protein (0.5 mg/ml), KCl (150 mM), MgCl2 (10 mM), sodium
isocitrate (8 mM), and isocitrate dehydrogenase (0.5 IU) in
Tris · HCl buffer (50 mM, pH 7.5). The mixtures
were preincubated for 5 min at 37°C, and the reaction was
started by addition of NADPH (1 mM). The incubation was carried out for
30 min at 37°C, and the reaction was terminated by acidifying to pH
3.5 with HCl. For outer medulla arachidonic acid metabolism, 15 µM
[1-14C]arachidonic
acid (0.2 µCi) and 0.25 mg/ml microsomal protein were used, and the
reaction was carried out for 45 min. Arachidonic acid and its
metabolites were extracted twice with ethyl acetate, and the combined
organic phase was washed once with double-distilled water. After
evaporation of organic solvent under nitrogen, the dry residue was
stored at 80°C until HPLC analysis.
HPLC analysis of arachidonic acid metabolites. A reverse-phase HPLC system consisting of a Shimadzu SLC-6A controller, two LC-6A pumps, and a Radiomatic 525TR flow scintillation analyzer with FLO-One software (Packard Instrument, Downers, IL) was used for the separation and quantification of arachidonic acid metabolites. Metabolites were separated on an Alltima C18 5 µm column (250 × 4 mm) with an Alltima C18 guard column and in-line filter (Alltech Associates, Deerfield, IL) exactly as described previously (13). Epoxygenase activity is reported as the sum of epoxide and dihydroxyeicosatrienoic acid formation.
In vitro inhibition of arachidonic acid metabolism. Microsomes prepared from the renal cortex or liver of untreated Sprague-Dawley rats (5 mg/ml) were incubated with varying concentrations of ABT, 1 mM NADPH, and an isocitrate dehydrogenase regenerating buffer system for 30 min at 37°C. An aliquot was removed immediately after the addition of NADPH as a control, and in some cases NADPH was omitted from the incubation mixture. After inactivation with ABT the microsome samples were diluted 10-fold with reaction buffer, and arachidonic acid metabolism was measured exactly as described previously.
Immunoblotting of CYP4A proteins. Renal cortical or hepatic microsomal protein (10 µg) or renal outer medullary microsomal protein (2 µg) was electrophoretically separated on a 8% SDS-polyacrylamide gel as described by Okita et al. (21). Proteins were transferred to a nitrocellulose membrane (Trans-Blot, Bio-Rad, Hercules, CA) in 25 mM Tris · HCl, 192 mM glycine, and 20% methanol. Membranes were incubated overnight with a 500-fold dilution of goat anti-CYP4A1 serum and for 2 h with a 1,000-fold dilution of alkaline phosphatase-conjugated rabbit anti-goat IgG (liver and cortex samples) or a 7,500-fold dilution of horseradish peroxidase-conjugated rabbit anti-goat IgG (outer medulla samples). Immunoreactive proteins were detected using an alkaline phosphatase conjugate substrate kit (Bio-Rad) or by enhanced chemiluminescence (Amersham) according to the manufacturer's instructions.
Statistics. Values are reported as means ± SE. Significance of difference between groups was evaluated by a paired t-test or a one-way ANOVA with post hoc multiple comparisons with a modified t-test. Statistical significance was set at the level of P < 0.05.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Inhibition of arachidonic acid metabolism by ABT. Although ABT has been widely used as a mechanism-based CYP inhibitor, its potency and selectivity for arachidonic acid metabolism were unknown. The inhibition of arachidonic acid metabolism by ABT was first measured in vitro. Renal cortical and hepatic microsomes from untreated Sprague-Dawley rats were incubated with varying concentrations of ABT in the presence of NADPH before their use in arachidonic acid metabolism determinations. As expected for a mechanism-based inhibitor, NADPH was required in the incubation mixture for inactivation of CYP enzymes. Arachidonic acid metabolism was inhibited by in vitro inactivation with ABT in both cortical and hepatic microsomes (Fig. 1). The inhibition was clearly dose dependent and showed a fair degree of selectivity for the CYP4A catalyzed formation of 19- and 20-HETE in the cortex. At a concentration of 500 µM ABT, cortical 19- and 20-HETE formation was reduced to 25-30% of control, whereas epoxygenase activity was reduced to 56% of control. In contrast, ABT showed no selectivity for the individual arachidonic acid metabolic pathways in the liver microsomes.
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-hydroxylation was inhibited at a dose of ABT as low as 5 mg/kg
and was reduced to 6% of control values with a 100 mg/kg dose. The
inhibition of 19-HETE formation by ABT showed a similar pattern as with
20-HETE, although the effect was less at all except the highest dose.
In contrast, arachidonic acid epoxide formation was not inhibited in
cortical microsomes except at an ABT dose of 100 mg/kg. At this dose
epoxygenase activity was reduced to 35% of control values. A similar
inhibition profile was found when arachidonic acid metabolism was
measured in outer medullary microsomes (Fig.
4B). The single major metabolite in
outer medulla microsomes is 20-HETE, with only minor amounts of 19-HETE
and the individual epoxides. As in the cortex, both 19- and 20-HETE
formations were reduced in a dose-dependent fashion in outer medulla
microsomes prepared from ABT-treated rats. Maximal inhibition of 19- and 20-HETE formation in the outer medulla was less than in the cortex
(60-70% inhibition), and epoxygenase activity was not inhibited
by ABT. In fact, at the 100 mg/kg dose of ABT, epoxygenase activity was
significantly increased in the outer medulla. The major CYP isoforms
responsible for arachidonic acid metabolism are CYP4A [- and
(-1)-hydroxylation], CYP2C23, CYP2E1, and CYP2J
[epoxidation and (-1)-hydroxylation] (15, 34). Thus, at
lower doses, ABT appears to show selectivity for the CYP4A enzymes in
the renal cortex and outer medulla. In contrast, ABT had a nonspecific
effect on hepatic arachidonic acid metabolism (Fig.
4C). Although only CYP4A enzymes
were inhibited at lower doses, as evidenced by the significant
inhibition of 20-HETE formation with as little as 5 mg/kg ABT, all of
the metabolic pathways were equally affected above 25 mg/kg.
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- and (-1)-hydroxylase activity in the cortex
and outer medulla was associated with loss of CYP4A immunoreactive
protein. In contrast, liver CYP4A protein levels remained constant
despite significant inhibition of functional activity. Two distinct
protein bands were detected in both cortex and liver microsomes from
control and treated animals. On the basis of the literature, the bottom band in the kidney samples can be identified as a doublet of CYP4A1 and
CYP4A2 and the upper band as CYP4A3. In the liver, the lower band is
CYP4A1 and the upper band is CYP4A3 (21). We were not able to detect
hepatic CYP4A2 in our samples, which reflects its low constitutive
levels in the liver and the cross-reactivity with the CYP4A1 antibody.
In the cortex, all three CYP4A isoforms showed parallel changes in
response to ABT.
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Recovery of functional activity after ABT treatment. The duration of the inhibitory response will be an important determinant of the effectiveness of mechanism-based inhibition to probe physiological function. The recovery of CYP content, arachidonic acid metabolism, and CYP4A protein levels was followed for 5 days after a single dose of ABT to Sprague-Dawley rats. The loss of cortical and hepatic microsomal CYP 6 h after administration of ABT was similar to that seen in the dose-response study (Fig. 6). CYP content showed signs of recovery within 48 h for the cortex and within 24 h for the liver. Recovery of CYP levels was faster in the liver, returning to basal levels by 72 h after ABT administration, whereas cortical CYP levels were significantly less than control values until 96 h after a single dose of ABT.
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- and (-1)-hydroxylase activity was maximally inhibited within 6 h after the ABT dose and gradually returned to basal levels over
3-4 days (Fig. 7). For
example, cortical 20-HETE formation was 17.9 ± 3.15% of control 6 h after ABT administration and returned to 84.8 ± 4.67% of control
by 72 h. In the cortex epoxygenase activity was only inhibited at the
6-h time point and was minimal (26% inhibition). Treatment with ABT
did not inhibit epoxygenase activity in the outer medulla. In the liver
the recovery of functional activity was identical for all three
pathways and was slower than recovery in the kidney. Hepatic
arachidonic acid epoxide and HETE formation was inhibited 60-76%
6 h after ABT administration and returned to basal values by 96 h.
Cortical and outer medullary CYP4A levels remained depressed until 72 h after a single dose of ABT (Fig. 8,
A and
B). Thus recovery of CYP4A protein
and arachidonic acid - and (-1)-hydroxylation in the kidney
showed identical patterns. In contrast, loss of CYP4A functional
activity in the liver was not due to a loss of CYP4A protein, as
evidenced by the constant level of CYP4A1 and CYP4A3 throughout the
120-h study period (Fig. 8C).
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Effect of ABT on blood pressure. ABT was administered to 7-wk-old SHRs to measure the effect of CYP inhibition on blood pressure. MAP was measured in freely moving animals through a catheter inserted into the abdominal aorta before and for up to 3 days after a single dose of ABT. Within several hours after treatment, MAP was significantly reduced and remained suppressed for over 12 h (Fig. 9). The effect was maximal within 4 h and averaged 17-23 mmHg during the 4- to 12-h period (8-24% decrease). ABT had no effect on heart rate during this period. Renal excretions of Na+ and K+, urine volume, and creatinine clearance were measured over the 24-h periods during the control and treatment phases of the protocol (Table 1). There was a 53% decrease in the 24-h urinary excretion of Na+ after a single dose of ABT. This is consistent with the decreased formation of 20-HETE, which normally promotes natriuresis and diuresis by its inhibitory effects on proximal tubular and medullary thick ascending limb of Henle Na+ transport. The effect of ABT on renal function and ion excretion was specific for Na+ because urinary K+ or creatinine excretion, creatinine clearance, and diuresis were unchanged.
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- and (-1)-hydroxylase activity in the SHR kidneys (Fig. 10).
20-HETE formation was inhibited 85% in the cortex and 70-80% in
the outer medulla during the 18-h period after ABT
administration. In contrast, cortical epoxygenase activity
was less inhibited than HETE formation and was unaffected in the outer
medulla. As expected from the previous studies, recovery of CYP
activity in the kidney was gradual. Within 72 h after ABT treatment,
arachidonic acid - and (-1)-hydroxylase activity recovered to
65-75% of control values in the cortex and to 75-85% of
control in the outer medulla. Epoxygenase activity was inhibited only
transiently and returned to basal levels within a day. The formation
rates of 19-HETE, 20-HETE, and EETs were similar in cortical microsomes
from Sprague-Dawley rats and SHRs. However, 19- and 20-HETE were
produced at 2.1- to 2.7-fold higher rates in the hypertensive outer
medulla microsomes than in the Sprague-Dawley microsomes. Outer medulla
epoxygenase activity was also significantly elevated (1.5-fold) in the
SHRs relative to the Sprague-Dawley rats. Total cortical CYP content
was reduced 50% in the 6- to 24-h samples and returned to control
values by 48 h after ABT treatment (data not shown). Measurement of
CYP4A protein levels by Western blotting showed that renal CYP4A
protein content was maximally reduced 8 h after ABT administration and
began to show signs of recovery by 48 h (data not shown). Complete
recovery to basal CYP4A protein levels was still not reached at 72 h,
indicating a slow CYP4A synthesis rate in the SHR kidney.
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Effect of 1-hydroxybenzotriazole on blood pressure. The 1-hydroxybenzotriazole compound is structurally very similar to ABT, having a single substitution of a hydroxyl group for the amino group at the one position of the triazole ring. In preliminary studies it was found that a single dose of 1-hydroxybenzotriazole had no effect on renal arachidonic acid metabolism. The effect of this compound on MAP was then investigated in the SHR. After a single dose of 1-hydroxybenzotriazole there was no significant change in MAP in the SHR (Fig. 11A). Treatment with 1-hydroxybenzotriazole also had no effect on Na+ excretion, creatinine excretion, or diuresis. Urinary excretion of K+ in the 1-hydroxybenzotriazole-treated rats increased 36% relative to the control period (Table 1). As expected from the preliminary studies, arachidonic acid metabolism was unaffected by treatment with this ABT analog (Fig. 11B). The fact that a structurally similar ABT analog has no effect on renal arachidonic acid metabolism or blood pressure provides strong evidence that ABT is modulating blood pressure through its effects on the renal CYP4A enzymes.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The characterization of ABT as an orally available mechanism-based inhibitor of renal arachidonic acid metabolism and the ability of almost complete inhibition of renal CYP4A activity to reduce blood pressure in the SHR are reported. The implication of CYP metabolites of arachidonic acid in the regulation of blood pressure is mostly supported by in vitro and in situ studies describing the effects of these eicosanoids on renal function and vascular tone (9, 23, 36, 37). Extension of these studies in vivo has been limited to the use of indirect inhibition of CYP enzymes by degradation of the prosthetic heme group or infusion of metabolically unstable CYP inhibitors into the medullary interstitium (16, 31, 32). ABT was first described as a mechanism-based CYP inhibitor in 1981, and since that time it has been used extensively both in vitro and in vivo in the characterization of hepatic CYP metabolism of xenobiotics (25, 27). We now show that ABT is an effective inhibitor of renal arachidonic acid metabolism in the rat cortex and outer medulla. ABT is metabolized by CYP enzymes to the reactive benzyne species, which then alkylates the prosthetic heme group of the enzyme (27). After an oral dose of ABT to rats tissue-to-plasma ratios were highest in tissues that contain relatively large amounts of CYP enzymes, namely the liver, kidney, and adrenals (33). In addition to the reactive benzyne metabolite, ABT is metabolized by N-acetylation and N-glucuronidation. The half-life of ABT and its metabolites in the plasma was found to be 9 h, whereas that in the kidney was 12 h. Less than 1% of a dose of ABT was accounted for in the kidney, suggesting that concentrations of ABT in the kidney necessary for inhibition of the CYP4A enzymes will be quite low.
ABT shows a striking selectivity for the CYP4A enzymes in both the kidney and the liver. In the liver this selectivity is evident only at very low doses (5-10 mg/kg), whereas in the kidney it persists at all except the highest dose of ABT. Maximal inhibition of arachidonic acid metabolism in the kidney is greatest for the CYP4A pathways, inhibiting 85% of 20-HETE formation and >70% of 19-HETE formation. ABT has generally been described as a nonselective inhibitor of hepatic CYP metabolism, implying that CYP enzymes with various active sites can accommodate this compound (25). The present results suggest that ABT can more easily enter the CYP4A active site than that of the arachidonic acid epoxygenases CYP2E1, CYP2C23, and CYP2J. Selectivity of ABT for the lung CYP4B enzymes has also been described (12).
The selectivity of ABT for the CYP4A enzymes is of particular
significance because other characterized CYP4A inhibitors cannot be
used in vivo. Substrates for the CYP4A enzymes are limited to fatty
acids and prostaglandins, which are hydroxylated preferentially at the
-position (15). The design of selective mechanism-based CYP4A
inhibitors has incorporated this relatively narrow substrate specificity. Acetylenic fatty acids access the CYP4A active site and
are enzymatically converted to a ketene species, which can alkylate the
protein (2). These acetylenic fatty acids inhibit lauric acid - and
(-1)-hydroxylation by hepatic microsomes and purified rat liver
CYP4A1 and PGE1 -hydroxylation
by CYP4A4 purified from pregnant rabbit lungs (2, 19, 29). They have
also been used in situ to establish the role of 20-HETE in the
regulation of renal blood flow and tubuloglomerular feedback and as a
K+ channel inhibitor in rat renal
arterioles (36-38). Both ABT and 10-undecynoic acid were used in
primary rat hepatocytes to demonstrate that CYP4A catalyzed fatty acid
metabolites mediate the induction of peroxisomal fatty acid
-oxidation and liver fatty acid binding protein by peroxisome
proliferators (11). Despite the selectivity of these acetylenic fatty
acid inhibitors for the CYP4A enzymes, their use is limited to in vitro
or in situ experiments because of their rapid degradation in vivo by
fatty acid -oxidation (29). One exception is an elegant study in
which 17-octadecynoic acid was chronically infused into the medullary
interstitium of rats and selectively inhibited CYP4A activity in the
outer medulla (32). The short half-life of this inhibitor is evident in
this study from the lack of effect on 20-HETE formation in the
neighboring cortex region. Based on our results, ABT can be
administered intraperitoneally and used to selectively inhibit CYP4A
enzymes in the rat kidney, providing an important tool for
characterizing the in vivo effects of 20-HETE. ABT can also be given
orally with a bioavailability of >70%, further increasing its
usefulness (33).
The inhibition of renal arachidonic acid metabolism was distinct from that measured in hepatic microsomes in several respects. Inhibition of 20-HETE formation was more complete in the cortex and outer medulla than in the liver, whereas epoxygenase activity was more susceptible to inhibition in the liver. One possible explanation for these findings is the relative abundance of the individual CYP isoforms in the liver versus the kidney. For example, three CYP4A genes (CYP4A1, CYP4A2, and CYP4A3) are expressed in the rat liver, whereas a fourth gene (CYP4A8) is also expressed in the rat kidney (15). The relative abundance of these isoforms also differs between the two tissues. In the liver, CYP4A1 and CYP4A3 are the major constitutive enzymes, whereas CYP4A2 is highly inducible. In contrast, in the kidney CYP4A2 and CYP4A8 are constitutively expressed at levels that are two to five times higher than that of CYP4A1 and CYP4A3 in the 7-wk-old rats used in these studies (13). A greater susceptibility of the CYP4A2 and CYP4A8 isoforms to ABT inhibition would account for the increased inhibition of 19- and 20-HETE formation in the kidney. This is consistent with the recent observation that ABT has no effect on the activity of recombinant CYP4A1 protein (5). The arachidonic acid epoxygenases CYP2C23, CYP2E1, and CYP2J are expressed in both the liver and kidney, and it is not clear which isoform is the major epoxygenase in these tissues (15, 34). On the basis of the present findings, it is likely that the major renal epoxygenase is more resistant to inhibition by ABT than is the major hepatic epoxygenase.
Surprisingly, renal CYP4A protein is lost following treatment with ABT, whereas hepatic CYP4A protein levels are unchanged. ABT has previously been shown to inactivate the CYPs by alkylation of the prosthetic heme moiety (27). The loss of CYP4A apoprotein in the cortex and outer medulla may reflect the smaller heme pool in the kidney relative to the liver. Assuming an insufficient pool to supply functional heme to the CYP4A apoprotein, it would be expected that the apoprotein would then be targeted for degradation. Alternatively, in the cortex and outer medulla ABT may also inhibit CYP4A enzymes at least in part by destruction of the apoprotein itself. Two distinct mechanisms of action may account for the increased CYP4A inhibition in the kidney relative to the liver. In the kidney the susceptible isoforms may also be more abundantly expressed, thereby accounting for the increased inhibition. Determination of the exact mechanism responsible for the loss of renal CYP4A apoprotein after ABT treatment will require further experimentation with the individual CYP4A recombinant proteins.
The decrease in MAP after ABT administration is the first report demonstrating that mechanism-based inhibition of CYP4A enzymes affects blood pressure in the SHR. Previous attempts at trying to associate renal arachidonic acid metabolism with the regulation of blood pressure have employed the administration of heme oxygenase inducers as an indirect method of altering CYP enzymes (16, 31). Induction of heme oxygenase reduces the levels of the prosthetic heme group and will therefore act as a nonspecific inhibitor of the CYP enzymes. In addition, heme oxygenase inducers may also affect vascular tone independent of their effects on the CYP enzymes because they alter the production of carbon monoxide and may interact with nitric oxide synthase. A single dose of ABT reduced blood pressure from 10 to 36 mmHg in individuals rats. MAP began to gradually decrease within 1 h after ABT treatment, and the effect was maximal by 4 h. This correlated with the rapid destruction of CYP enzymes and inhibition of arachidonic acid metabolism, which was also maximal within 4 h. The observation that a structurally similar noninhibitory ABT analog has no effect on blood pressure provides strong evidence that the decrease in blood pressure after ABT administration is associated with its potent inhibitory effects on 20-HETE formation.
A decrease in blood pressure after CYP inhibition of arachidonic acid metabolism is consistent with some of the prohypertensive properties of the CYP4A eicosanoids and with the changes in renal function, vascular tone, and CYP4A expression that occur in the SHR. For example, 20-HETE is a potent vasoconstrictor, depolarizes vascular smooth muscle cells, and inhibits Na+-K+-ATPase (10, 30, 36). An increased production of 20-HETE has been proposed to alter renal vascular tone and elevate blood pressure in the SHR, whereas a decreased production of 20-HETE such as that observed after ABT treatment would be expected to reduce the vasoconstrictive effects and lower blood pressure. In contrast, the effects of 20-HETE on Na+ transport in the renal tubule result in natriuresis and diuresis, and a dampening or elimination of these effects resulting from decreased 20-HETE formation could lead to an increase in blood pressure. Inhibition of renal 20-HETE formation by ABT produced the expected decrease in urinary Na+ excretion, but this was not associated with an increase in blood pressure as might be predicted. This suggests that the effects of 20-HETE in regulating renal vascular tone are more important in the regulation of blood pressure than its effects on tubular Na+ transport.
The importance of 20-HETE in regulating vascular tone and blood
pressure is also supported by previous studies. Vascular tone is
altered very early in the SHR, with basal diameters of afferent arterioles and preglomerular vasculature 18-35% smaller in the SHR than in the normotensive WKY rat (8). Addition of CYP inhibitors to
the perfusate of juxtamedullary microvascular preparations completely
eliminated these differences in vascular tone, indicating that CYP
metabolites of arachidonic acid are important determinants of these
changes. An increased formation of 20-HETE has also been reported in
renal microsomes from young SHRs relative to normotensive WKY rats (8,
13, 22). Our recent studies suggest that increased expression of CYP4A3
and CYP4A8 in the young SHR kidney is responsible for the increased
20-HETE formation (13). However, the significance of this increased
CYP4A -hydroxylase activity in the elevated blood pressure in the
SHR is still unclear because the absolute levels of cortical activity
are similar in other normotensive rat strains, including the
Sprague-Dawley rats used in this study and Lewis rats (8). Despite
similar levels of 20-HETE formation in the SHR and other normotensive
rats, it is possible that the SHR vasculature may be more responsive to
its vasoconstrictive effects, thus accounting for the differences in
blood pressure between these animals. In the case of the WKY rat a
difference in vascular responsiveness would be combined with a
decreased production of 20-HETE.
The overall effect of the CYP eicosanoids on renal function and blood pressure will be dependent on the relative production of the individual metabolites within specific regions of the nephron. Because the CYP4A enzymes are more potently inhibited by ABT than the other arachidonic acid metabolizing CYP enzymes, the EETs become quantitatively more important metabolites in renal microsomal fractions from the treated animals. In SHR cortical microsomes the percentage of total arachidonic acid metabolism that goes through the epoxygenase pathway increases from 45% in control animals to 71-76% during the 24-h period after ABT administration. The increase in EET contribution could be completely accounted for by a decrease in the contribution of 20-HETE. Assuming that the in vitro metabolite formation reflects the in vivo formation, then the antihypertensive properties of the EETs may become dominant after CYP4A inhibition. The inhibitory effects of 5,6- and 11,12-EET against Na+-K+-ATPase, and the vascular smooth muscle cell hyperpolarization and ensuing vasodilation by 11,12- and 14,15-EET would lead to a lowered blood pressure (9, 23).
A persistent suppression of CYP4A functional activity was not sufficient to maintain a decreased blood pressure in the SHR. A single dose of ABT inhibits functional CYP4A activity in the cortex for >72 h, whereas MAP is completely recovered 18-24 h after treatment. Several explanations could account for this observation. First, the return of blood pressure to basal levels may be a physiological response that does not involve the CYP renal eicosanoids and that could supersede any effects of these metabolites on renal function and vascular tone. Arterial blood pressure is normally tightly controlled, owing to the multiple levels of regulation (7). Pressure controls that act on neural receptors respond within seconds to changes in blood pressure, followed by activation of hormonal control systems within minutes. The kidney-fluid system is necessary for long-term control of arterial pressure and reacts within hours or days of blood pressure changes. The return of blood pressure toward control values within 12-24 h after ABT treatment suggests that the kidney-fluid system may be operating in this case.
A second possibility that could account for the discrepancy between the duration of blood pressure suppression and inhibition of 20-HETE formation is that CYP epoxygenase activity is the more important determinant of blood pressure in the SHR. Epoxygenase activity was transiently inhibited after administration of ABT, although the effect was much less than the corresponding inhibition of CYP4A activity and 20-HETE formation. It also returned to control values within 12 h, suggesting that ABT was not inhibiting the arachidonic acid epoxygenases in a mechanism-based manner. Although MAP and cortical arachidonic acid epoxygenase activity recovered in a similar time frame, a direct reduction of blood pressure by inhibition of EET formation is not supported by the biological properties of these metabolites. In general, the EETs are considered to be antihypertensive because of their vasodilatory effects and ability to inhibit Na+-K+-ATPase in the tubular epithelium (9, 23). One exception is the 5,6-EET metabolite, which has vasoconstrictive properties (9). It is possible that the decrease in blood pressure could be a result of the increased contribution of the antihypertensive epoxide metabolites to the overall metabolism of arachidonic acid at early times after ABT administration. However, the EETs remain the major metabolic product until 72 h after ABT administration, which is also not consistent with the return of blood pressure to basal levels.
A final explanation that can account for the discrepancy in the
recovery of blood pressure and CYP4A activity is that in vitro determinations of enzyme activity do not reflect the in vivo formation of 20-HETE. CYP4A inhibition by ABT involves destruction of the enzymes
and requires synthesis of new protein for recovery. After a single dose
of ABT, complete recovery of CYP4A functional activity required >72
h, indicating a slow synthesis rate of the protein. Even longer
recovery periods were reported for lauric acid -hydroxylase activity
in hepatic microsomes after a 50 mg/kg dose of ABT to Sprague-Dawley
rats (26). Despite this slow recovery it is possible that sufficient
activity to provide basal levels of 20-HETE exists before complete
recovery of CYP4A activity. It was recently suggested that 20-HETE can
also be stored in the phospholipid pool and released in response to ANG
II stimulation (4). This raises the interesting possibility that
20-HETE may be available at sufficient concentrations to produce a
desired effect despite very low levels of functional CYP4A protein.
Quantification of endogenous levels of the CYP eicosanoids in the renal
cortex and outer medulla will provide a more accurate measurement of
the functional levels of these metabolites.
In summary, we have characterized the mechanism-based CYP inhibitor ABT as a selective inhibitor of the renal CYP4A enzymes. Inhibition of the CYP4A enzymes occurs rapidly and leads to loss of apoprotein and a decreased formation of 19- and 20-HETE formation in microsomes prepared from the outer medulla and cortex. Recovery of enzyme activity involves the synthesis of new protein and occurs over >3 days. Administration of ABT to SHRs results in inhibition of up to 85% of renal 20-HETE formation and is accompanied by an acute reduction in blood pressure. The effects of ABT on blood pressure appear to be specific to its CYP4A- inhibitory properties because a structurally similar analog that does not inhibit 20-HETE formation has no effect on blood pressure. Finally, the decrease in blood pressure with ABT treatment is not consistent with changes in renal tubular ion transport or gross changes in renal function, suggesting that the vasoconstrictive properties of 20-HETE are a major determinant of its effect on blood pressure.
Perspectives
The present studies were carried out in the SHR during the developmental period of hypertension. Although these results further support a role for CYP eicosanoids in the regulation of blood pressure, the mechanism by which this occurs remains unclear. It is not known whether 20-HETE plays a role in elevating blood pressure only in rats genetically prone to develop hypertension or if it is also an important factor in normal blood pressure control. Preliminary studies suggest that inhibition of 20-HETE formation also reduces blood pressure in the normotensive Sprague-Dawley rat. The relative importance of 20-HETE in the prehypertensive stage of the disease compared with the developmental and established phases is also not known. It will be important to examine whether early modulation of the CYP4A enzymes can prevent the development of hypertension and whether it can reverse the elevated blood pressure in older animals with established disease. The vascular changes that accompany CYP inhibition also need to be characterized. Further examination of the distinct roles for 20-HETE in the regulation of blood pressure in the SHR and the Dahl salt-sensitive models of hypertension will increase our understanding of the diverse biological properties of this eicosanoid. The availability of an orally available mechanism-based CYP inhibitor with a high degree of selectivity for the renal CYP4A enzymes provides a useful tool for addressing these questions. Further characterization of the role of 20-HETE and other CYP eicosanoids in the regulation of renal function and blood pressure will provide the framework for extension of this work into understanding the role of CYP catalyzed arachidonic acid metabolism in human essential hypertension.| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Zhigang Yu for assistance with image analysis, the laboratory of Dr. R. Curtis Morris, Jr., for assistance with the catheterization technique, and Joan H. Ottaway and Janice Yuan in the General Clinical Research Center for clinical chemistry measurements.
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FOOTNOTES |
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This work was supported in part by the National Heart, Lung, and Blood Institute Grant HL-53994. P. Su was supported in part by the University of California Toxic Substances Research and Teaching Program.
Address for reprint requests: D. L. Kroetz, Dept. of Biopharmaceutical Sciences, 513 Parnassus, Box 0446, San Francisco, CA 94143-0446.
Received 30 October 1997; accepted in final form 15 April 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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K. Nakagawa, J. S. Marji, M. L. Schwartzman, M. R. Waterman, and J. H. Capdevila Androgen-mediated induction of the kidney arachidonate hydroxylases is associated with the development of hypertension Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2003; 284(4): R1055 - R1062. [Abstract] [Full Text] [PDF]
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D. E. Stec, A. Flasch, R. J. Roman, and J. A. White Distribution of cytochrome P-450 4A and 4F isoforms along the nephron in mice Am J Physiol Renal Physiol, January 1, 2003; 284(1): F95 - F102. [Abstract] [Full Text] [PDF]
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F. Xu, W. O. Straub, W. Pak, P. Su, K. G. Maier, M. Yu, R. J. Roman, P. R. Ortiz De Montellano, and D. L. Kroetz Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid omega -hydroxylase activity Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2002; 283(3): R710 - R720. [Abstract] [Full Text] [PDF]
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M. Alonso-Galicia, K. G. Maier, A. S. Greene, A. W. Cowley Jr., and R. J. Roman Role of 20-hydroxyeicosatetraenoic acid in the renal and vasoconstrictor actions of angiotensin II Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2002; 283(1): R60 - R68. [Abstract] [Full Text] [PDF]
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M.-H. Wang, B. A. Zand, A. Nasjletti, and M. Laniado-Schwartzman Renal 20-hydroxyeicosatetraenoic acid synthesis during pregnancy Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2002; 282(2): R383 - R389. [Abstract] [Full Text] [PDF]
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 R. J. Roman P-450 Metabolites of Arachidonic Acid in the Control of Cardiovascular Function Physiol Rev, January 1, 2002; 82(1): 131 - 185. [Abstract] [Full Text] [PDF]
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F. Zhang, M.-H. Wang, U.M. Krishna, J. R. Falck, M. Laniado-Schwartzman, and A. Nasjletti Modulation by 20-HETE of Phenylephrine-Induced Mesenteric Artery Contraction in Spontaneously Hypertensive and Wistar-Kyoto Rats Hypertension, December 1, 2001; 38(6): 1311 - 1315. [Abstract] [Full Text] [PDF]
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 I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
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B. Lopez, C. Moreno, M. G. Salom, R. J. Roman, and F. J. Fenoy Role of guanylyl cyclase and cytochrome P-450 on renal response to nitric oxide Am J Physiol Renal Physiol, September 1, 2001; 281(3): F420 - F427. [Abstract] [Full Text] [PDF]
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S. I. Pomposiello, M. A. Carroll, J. R. Falck, and J. C. McGiff Epoxyeicosatrienoic Acid-Mediated Renal Vasodilation to Arachidonic Acid Is Enhanced in SHR Hypertension, March 1, 2001; 37(3): 887 - 893. [Abstract] [Full Text] [PDF]
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E. R. Jacobs and D. C. Zeldin The lung HETEs (and EETs) up Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H1 - H10. [Abstract] [Full Text] [PDF]
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Z. Yu, F. Xu, L. M. Huse, C. Morisseau, A. J. Draper, J. W. Newman, C. Parker, L. Graham, M. M. Engler, B. D. Hammock, et al. Soluble Epoxide Hydrolase Regulates Hydrolysis of Vasoactive Epoxyeicosatrienoic Acids Circ. Res., November 24, 2000; 87(11): 992 - 998. [Abstract] [Full Text] [PDF]
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M. M. Muthalif, N. A. Karzoun, L. Gaber, Z. Khandekar, I. F. Benter, A. E. Saeed, J.-H. Parmentier, A. Estes, and K. U. Malik Angiotensin II-Induced Hypertension : Contribution of Ras GTPase/Mitogen-Activated Protein Kinase and Cytochrome P450 Metabolites Hypertension, October 1, 2000; 36(4): 604 - 609. [Abstract] [Full Text] [PDF]
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H. Honeck, V. Gross, B. Erdmann, E. Kargel, R. Neunaber, A. F. Milia, W. Schneider, F. C. Luft, and W.-H. Schunck Cytochrome P450-Dependent Renal Arachidonic Acid Metabolism in Desoxycorticosterone Acetate-Salt Hypertensive Mice Hypertension, October 1, 2000; 36(4): 610 - 616. [Abstract] [Full Text] [PDF]
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K. G. Maier, L. Henderson, J. Narayanan, M. Alonso-Galicia, J. R. Falck, and R. J. Roman Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H863 - H871. [Abstract] [Full Text] [PDF]
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M. M. Muthalif, I. F. Benter, Z. Khandekar, L. Gaber, A. Estes, S. Malik, J.-H. Parmentier, V. Manne, and K. U. Malik Contribution of Ras GTPase/MAP Kinase and Cytochrome P450 Metabolites to Deoxycorticosterone-Salt-Induced Hypertension Hypertension, January 1, 2000; 35(1): 457 - 463. [Abstract] [Full Text] [PDF]
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