Department of Pharmacology, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
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20-Hydroxyeicosatetraenoic acids (20-HETE), a
biologically active cytochrome P-450
(CYP) metabolite of arachidonic acid in the rat kidney, can be
catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To
determine the contribution of CYP4A isoforms to renal 20-HETE
synthesis, specific antisense oligonucleotides (ODNs) were developed,
and their specificity was examined in vitro in Sf9 cells
expressing CYP4A isoforms and in vivo in Sprague-Dawley rats.
Administration of CYP4A2 antisense ODNs (167 nmol · kg body wt1 · day
1
iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no
effect on tubular synthesis, whereas administration of CYP4A1 antisense
ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%,
respectively. RT-PCR of microdissected renal microvessel RNA indicated
the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a
CYP4A1-immunoreactive protein was detected by Western analysis of
microvessel homogenates. Blood pressure measurements revealed a
reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1
as a major 20-HETE synthesizing activity in the rat kidney and further
document the feasibility of using antisense ODNs to specifically
inhibit 20-HETE synthesis and thereby investigate its role in the
regulation of renal function and blood pressure.
arachidonic acid; antisense oligonucleotides; preglomerular microvessels; proximal tubules; blood pressure
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INTRODUCTION |
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PREVIOUS STUDIES HAVE established that
20-hydroxyeicosatetraenoic acid (20-HETE), the -hydroxylation
product of arachidonic acid, is the principal arachidonic acid
metabolite formed in tubular and vascular structures of the rat renal
cortex and outer medulla (11, 22). The synthesis of 20-HETE is age
dependent and is higher in kidneys obtained from spontaneously
hypertensive rats (21). Numerous studies have demonstrated that 20-HETE
is endowed with potent biological activities and have provided evidence
that it contributes to the regulation of renal vascular and tubular functions and to the control of arterial pressure (18, 20, 35, 36).
The -hydroxylation of fatty acids, including arachidonic acid, is
catalyzed by enzymes of the CYP4A family. In the rat, four isoforms
have been identified: CYP4A1, CYP4A2, CYP4A3, and CYP4A8 (14, 15). Messages for all four of these isoforms have been identified
in the kidney (28). These isoforms, although sharing 66-98%
homology and a common catalytic activity, i.e., hydroxylation of fatty
acids at the
-carbon, are localized to different renal structures
and are exposed to different regulatory mechanisms. For example,
whereas CYP4A2 is preferentially expressed in the outer-medullary/thick
ascending limb of Henle's loop region, CYP4A1 and CYP4A3 are highly
expressed in the proximal tubules (9, 13). In addition, CYP4A2 is
believed to be the major CYP4A isoform expressed in the renal
microvasculature, a major site of 20-HETE synthesis and action (11).
Renal CYP4A1 and CYP4A3 can be induced by hypolipidimic drugs such as
clofibrate, whereas CYP4A2 is thought to be constitutively expressed,
especially in male rats (10, 14, 15, 29). The renal expression of these
isoforms is age dependent; CYP4A1 and CYP4A3 proteins are detectable in
the fetus, and their levels gradually increase from birth until ~9 wk
of age, followed by a progressive decline to very low levels in adults. CYP4A2 protein is undetectable until 5 wk, but then the levels increase
so that in adult male rats it is the major isoform detected in the
kidney (16, 17).
The role of each of the CYP4A proteins in the generation of endogenous
20-HETE in the rat kidney is yet to be determined. Studies with
purified and recombinant proteins characterize some of the catalytic
activities and substrate specificities of the rat CYP4A isoforms (1, 3,
7, 27, 30). Recent studies in our laboratory, using
baculovirus-expressed recombinant CYP4A proteins, demonstrated that
CYP4A1, CYP4A2, and CYP4A3, but not CYP4A8, exhibit significant
arachidonic acid -hydroxylating activity. Moreover, CYP4A2 and
CYP4A3, which share 97% homology, have similar catalytic properties,
both catalyzing arachidonic acid
-hydroxylation and
11,12-epoxidation. In contrast, CYP4A1 functions solely as an
arachidonate
-hydroxylase with a turnover rate 20 times higher than
that of CYP4A2 or CYP4A3 (unpublished data). Because of the high
sequence homology among CYP4A isoforms, overlapping catalytic activities, and the similar sensitivity to metabolic inhibitors, it is
difficult to evaluate the contribution of each of these isoforms to the
synthesis of 20-HETE in the kidney.
Antisense oligonucleotides (ODNs) have been widely used as specific inhibitors of gene expression. They offer the possibility of blocking the expression of a particular gene without any changes in the function of other genes. Several investigators used antisense ODNs to inhibit specific CYP activities in vitro and in vivo with significant success (5, 32). In the present study, we designed antisense ODNs against CYP4A1 and CYP4A2/4A3 expression and evaluated their effectiveness for achieving isoform-specific inhibition of 20-HETE synthesis in vitro and in vivo.
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METHODS |
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Materials.
[1-14C]arachidonic
acid (56 mCi/mmol) was obtained from DuPont-New England Nuclear
(Boston, MA). Purified recombinant rat NADPH-cytochrome
P-450
(c) oxidoreductase (OR; EC
1.6.2.4) (specific activity, 58 µmol · min1 · mg
1)
was purchased from Oxford Biomedical Research (Oxford,
MI). Emulgen E911 was obtained from KAO Atlas (Tokyo,
Japan). Tissue culture and molecular biology reagents were purchased
from Life Technologies (Gaithersburg, MD). Sf9
insect cells and the liposome DNA transfection kit were from Invitrogen
(San Diego, CA). Purified recombinant rat liver cytochrome
b5 (specific
activity, 40 nmol/mg) was purchased from Panvera (Madison,
WI). All solvents were HPLC grade.
Preparation of recombinant CYP4A cell
membranes. CYP4A proteins were expressed in the
baculovirus-Sf9 insect cell expression system as described
previously (33). CYP4A recombinant Sf9 cell membranes were
prepared after infection with the recombinant virus and incubation in
the presence of hemin (4 µg/ml) for 72 h followed by centrifugation
at 100,000 g for 60 min of cell
lysates as described (33). The membrane pellets were
resuspended in sucrose buffer (50 mM potassium phosphate, pH 7.4, and
0.5 M sucrose) and stored at 80°C. Protein concentration was
determined according to the method of Bradford (Bio-Rad, Melville, NY).
Cytochrome P-450 content was
calculated from the reduced CO-difference spectrum using
an extinction coefficient of 91 mM
1 (23).
Preparation and selection of ODNs.
Antisense and scrambled ODNs were targeted to bases 3 to +21 of
the CYP4A1 and CYP4A2 cDNAs (15) encompassing the ATG translation site
codon. The scrambled ODNs were designed to have the same base
composition as the antisense ODNs. All ODN sequences were aligned to
the DNA database (GenBank) using the MacVector Sequence Analysis
Software. The phosphorothioate derivatives of the selected ODNs were
synthesized and purified by Gene Link (Thornwood, NY). The following
ODNs were used: antisense for CYP4A2 (4A2-AS),
5'-GCT-AAA-TAC-AGA-GAA-ACC-CAT-GGT-3'; scrambled ODN for
CYP4A2 (4A2-S), 5'-CAG-ACC-GCA-GGA-CTA-AAT-AGA-TAT-3'; antisense for CYP4A1 (4A1-AS),
5'-CAG-TGC-AGA-GAC-GCT-CAT-GGT-3' (21 bases) and
5'-CAG-TGC-AGA-GAC-GCT-CAT-3' (18 bases); scrambled ODN for
CYP4A1 (4A1-S), 5'-CTG-ACC-GCA-GCA-GGA-CTT-AGA-TGG-3'. The
potency and specificity of antisense ODNs were tested in the baculovirus/Sf9 cell system. Briefly, Sf9 cells (1 × 105 cells/dish) were
infected with CYP4A recombinant viruses in the presence of hemin (4 µg/ml). Various concentrations of liposome-encapsulated antisense
ODNs, scrambled ODNs, or liposome alone were added into 3-cm culture
dishes at 3, 24, 48, and 72 h after infection. The amount of liposomes
was fixed at 1 µg per each 0.06 nmol of ODNs. Twenty-four hours after
the last treatment, cells were harvested, and membranes were prepared
for analyses.
Preparation of liposomes. Cationic
liposomes composed of dimethyldioctadecylammonium bromide and
L--dioleylphosphatidylethanolamine (2:5, wt/wt; Avanti Polar Lipid, Alabaster, AL) were dissolved in 1 ml
chloroform, and the solvent was evaporated under
N2 gas at room
temperature. Liposomes were prepared by resuspending the lipids in 9 ml
of sterile deionized water and sonication on ice until the solution was
almost clear (19).
Animal treatment. Male Sprague-Dawley
rats (Harlan, 7 wk old; n = 4 per
group) anesthetized with pentobarbital sodium (60 mg/kg ip) were
instrumented with two polyethylene (PE-50) catheters. One catheter was
placed into the femoral artery for blood pressure measurement, and the
other one was placed into the femoral vein for administration of ODNs.
The rats were allowed 24 h to recover before commencing treatment.
Liposome-encapsulated antisense ODNs or scrambled ODNs (1 µg/0.06
nmol ODNs) were injected daily as a bolus at doses of 167 nmol · kg body
wt1 · day
1
for 5 days. Additional rats were treated with liposomes mixed with the
ODN vehicle (water). In some experiments, rats were injected with two
antisense preparations, in combination, each at a dose of 100 nmol · kg body
wt
1 · day
1.
On day 6, after the onset of
treatment, the rats were killed, and tissues (liver and kidney) were
removed for preparation of microsomes and isolation of preglomerular
vessels and proximal tubules as previously described (33, 34). The
purity of each preparation was ascertained by phase-contrast microscopy
and by the presence or absence of immunoreactive
-glutamyl
transpeptidase (
-GT), a specific marker for the proximal tubule
brush border (2). The microvessel preparation was estimated to be 98%
pure by phase-contrast microscopy (Fig.
1A).
Furthermore, Western analysis of microvessel homogenates with antibody
against
-GT revealed no immunoreactive protein in the microvessel
preparation. In contrast, proximal tubular homogenates showed a strong
immunoreactive band (Fig. 1B). To
examine the specificity of these ODNs, CYP2E1-derived p-nitrophenol hydroxylation and
cytochrome P-450
(c) reductase activity were measured
as previously described (33).
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Arachidonic acid metabolism. Microsomes prepared from different tissues (150 µg) were preincubated with [1-14C]arachidonic acid (0.4 µCi, 7 nmol) in 100 mM potassium phosphate buffer, pH 7.4, containing 10 mM MgCl2 for 3 min at 37°C. NADPH (1 mM) and the reaction mixture with a final volume of 0.4 ml was incubated for 30 min at 37°C. For recombinant CYP4A proteins, CYP4A-expressed Sf9 cell membranes (10 pmol P-450) were mixed with purified OR and cytochrome b5 at a molar ratio of 1:14:4 in a final volume of 150 µl buffer and incubated for 3 min on ice followed by incubation with [1-14C]arachidonic acid (0.4 µCi, 7 nmol) for an additional 3 min before adding NADPH (1 mM). Incubations were carried out for 30 min at 37°C. The reaction was terminated by acidification to pH 3.5-4.0 with 2 M formic acid, and metabolites were extracted with ethyl acetate. The final extract was evaporated under nitrogen, resuspended in 50 µl of methanol, and injected onto the HPLC column. Reverse-phase HPLC was performed on a 5 µm ODS-Hypersil column, 4.6 × 200 mm (Hewlett-Packard, Palo Alto, CA) using a linear gradient ranging from acetonitrile:water:acetic acid (50:50:0.1) to acetonitrile:acetic acid (100:0.1) at a flow rate of 1 ml/min for 30 min. The elution profile of the radioactive products was monitored by a flow detector (In/us System, Tampa, FL). The identity of each metabolite was confirmed by its comigration with an authentic standard.
Western blot analysis. Goat anti-rat
CYP4A1 antibody was purchased from Gentest (Woburn, MA). Although this
polyclonal antibody preparation cross-reacts with all CYP4A proteins,
its sensitivity for detecting CYP4A1 protein is ~30 times higher than
that of CYP4A2 protein and 2 and 9 times greater than CYP4A3 and CYP4A8 proteins, respectively. CYP4A-expressed Sf9 cell membranes were separated by electrophoresis on an 8% SDS-polyacrylamide gel at 25 mA/gel at 4°C for 18-20 h, and immunoblotting using either phosphoimaging or alkaline phosphatase detection systems was performed. Microsomes prepared from microvessels and proximal tubules were treated
with SDS reducing buffer containing 62.5 mM
Tris · HCl, pH 6.8, 10% glycerol, 2%
SDS, 5% -mercaptoethanol, and 0.125% (wt/vol)
bromophenol blue, then further incubated in boiling water for 4 min.
SDS gel electrophoresis was performed, and immunoreactive CYP4A
proteins were detected as described above. Antibody against
-GT was
kindly provided by Dr. R. P. Hughey, University of Pittsburgh Medical
School. Western analysis for
-GT was performed as described previously (2).
RT-PCR. Renal microvessels (3 mm in
length) were microdissected and connective tissue was removed under
microscope. The microvessels were transferred by
microbeads (0.5-1 mm in diameter) into 10 µl of a
buffer containing 2% (vol/vol) Triton X-100, 5 mM DTT, and 1,600 U/ml
of RNasin (Promega, Madison, WI). The microvessels were
immediately frozen on dry ice and stored in 80°C until
RT-PCR reaction was performed. The total RNA of the whole kidney was extracted using TRIzol reagent (Life Technologies). The
RNA pellet was resuspended in nuclease-free water. A reverse
transcription reaction was performed using a First-strand cDNA
synthesis kit (Pharmacia Biotech, Milwaukee, WI). Briefly, RNA (3 µg)
from whole kidney or 10 µl of microvessel permeability solution was
added to 15 µl reverse transcription reaction mixture containing 45 mM Tris (pH 8.3), 68 mM KCl, 15 mM DTT, 9 mM
MgCl2, 0.08 mg/ml BSA, 1.8 mM
dNTP, 40 pmol of either CYP4A1, 4A2, or 4A3 backward primer, and
Moloney murine leukemia virus reverse transcriptase. The reactions were
incubated for 1 h at 37°C and terminated by heating to 95°C for
5 min. PCR was carried out in a 100-µl reaction mixture containing 50 mM Tris · HCl (pH 9.0), 20 mM
NH4SO4,
3 mM MgCl2, 200 µM dNTPs, 20 pmol of specific CYP4A1, 4A2, and 4A3 primer pairs, and 10 µl of
first strand synthesis reaction from whole kidney or microvessels. PCR
was also performed using the CYP4A plasmids (100 ng) as a template
for positive controls. Samples containing Ampliwax
(Perkin-Elmer, Branchburg, NJ) were denatured by heating to 80°C
for 8 min, cooled to 2°C for 2 min, and heated to 22°C for 2 min to solidify the wax. Tf1 DNA polymerase (1 U; Epicentre
Technologies, Madison, WI) was added to initiate a "hot-start" reaction. Reactions were cycled 30 times through a 1-min denaturing step at 95°C, a 1-min annealing step at 50°C, and a 1-min
extension step at 70°C. After the cycling procedure, a final 10-min
elongation step at 70°C was performed. The specific CYP4A1, 4A2,
and 4A3 primers were designed to amplify 827-, 317-, and 321-bp
fragments from each of the corresponding cDNAs. Additional nested PCR
amplification was performed for CYP4A1 (300-bp expected fragment size).
The sequences of the primers used were as follows: CYP4A1, 5'-GTA TCC AAG TCA CAC TCT CCA-3' (forward primer), 5'-CAG GAC ACT
GGA CAC TTT ATT G-3' (backward primer for 827 bp), 5'-CAG
AGG TGT TTG ACC CTT CCA G-3' (forward primer, nested PCR),
5'-CCA TCA AGG TTG TGA CCG TTG-3' (backward primer, nested
PCR for 300 bp); CYP4A2, 5'-AGA TCC AAA GCC TTA TCA ATC-3'
(forward primer), 5'-CAG CCT TGG TGT AGG ACC
T-3' (backward primer); CYP4A3, 5'-CAA AGG CTT CTG GAA TTT ATC-3' (forward primer), 5'-CAG CCT TGG TGT
AGG ACC T-3' (backward primer). An aliquot (12 µl) of each PCR
reaction was separated by 1.5% agarose gel, and PCR products were
stained with ethidium bromide. The PCR products were transferred from the agarose gel to nylon membranes (Hybond-N, Amersham)
by the capillary method for 18 h, ultraviolet cross-linked to the
membrane, and prehybridized for 30 min at 65°C in a
rapid-hybridization buffer (Amersham). Southern hybridization was
performed using 32P-labeled CYP4A
cDNA probes (Rediprimer Kit, Amersham).
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RESULTS |
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Antisense ODNs were designed to specifically recognize the ATG region
of the CYP4A isoforms. The 4A2-antisense ODN contained 24 nucleotides
and by computer analysis showed no homology to CYP4A1 or CYP4A8 mRNA
sequences. However, it did recognize CYP4A3 mRNA; the homology between
these two isoforms in their coding regions is 97%. The 4A2-scrambled
ODN contained the same base composition and showed by computer analysis
no sequence homology with CYP4A2 or any known CYP sequences. The
specificity and potency of these ODNs on CYP4A2 protein levels and
CYP4A2-derived arachidonic acid -hydroxylation were assessed in the
Sf9 cells expressing CYP4A2. As seen in Fig.
2A,
20-HETE synthesis in Sf9 cells treated with 0.1 µM 4A2-antisense ODN
was inhibited by 72%. At high concentrations of 4A2-antisense ODN (1 and 10 µM), arachidonate
-hydroxylase activity was decreased to
~20% of control. The 4A2-scrambled ODN had little effect on
CYP4A2-catalyzed 20-HETE synthesis at 0.1 and 1 µM. However, at high
concentration (10 µM) the specificity disappeared; 4A2-scrambled ODN
caused ~50% inhibition of 20-HETE synthesis.
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With respect to CYP4A1, two different lengths of antisense ODNs were
examined for their potency and specificity in the baculovirus-Sf9 cell
system. Both were targeted to the ATG initiation codon of the CYP4A1
cDNA; one contained 18 nucleotides and the other 21 nucleotides. The
results depicted in Fig. 2B indicate
that both antisense ODNs inhibited arachidonate -hydroxylation in a
concentration-dependent manner. However, the 21-mer antisense ODN was
more potent, inhibiting 20-HETE synthesis by 75% at 10 µM. The
4A1-scrambled ODN had little effect on 20-HETE synthesis, inhibiting it
by 20% at 10 µM (Fig. 2B).
Additional control experiments revealed no effect of the liposome
vehicle on 20-HETE synthesis. Moreover, treatment of cells expressing
CYP4A1 protein with 4A2-AS or of cells expressing CYP4A2 with 4A1-AS
did not affect 20-HETE synthesis (data not shown).
The specific antisense ODNs were used for evaluating whether selective
inhibition of CYP4A-derived 20-HETE synthesis can be achieved in vivo.
The dosage of the ODN preparations (phosphorothioate derivatives mixed
with liposomes for increased efficiency of delivery and uptake) used
for the in vivo studies was arbitrarily chosen; it was primarily based
on literature research and partially on our in vitro screening. Figure
3 contrasts the effects of in vivo treatment of 4A2- and 4A1-antisense ODNs on 20-HETE synthesis by
microvessels and proximal tubules homogenates. We used the 21-base
antisense ODN (4A1-AS) for the in vivo experiments. Rats were treated
with 4A2- or 4A1-antisense ODNs for 5 days at 167 nmol · kg1 · day
1.
Both 4A2- and 4A1-antisense ODNs significantly decreased 20-HETE synthesis in microvessel homogenates by 40 and 50%, respectively. Proximal tubular synthesis of 20-HETE was markedly inhibited by 50% in
rats treated with 4A1-antisense ODN but was unaffected in rats treated
with 4A2-antisense ODN. In two additional sets of experiments, rats
were treated with both antisense preparations (each ODN preparation at
100 nmol/kg) following the same protocol. The combined administration
of both antisense preparations resulted in a significant 75%
inhibition of renal microvessel 20-HETE synthesis. Additional analysis
revealed that 20-HETE synthesis was also inhibited in liver microsomes
by ~50% in rats treated with the CYP4A antisense preparations. For
example, 4A2-antisense ODN reduced 20-HETE synthesis from 134 ± 4 to 67 ± 1 pmol · mg
1 · min
1,
whereas the 4A2-scrambled ODN reduced it to 108 ± 6 pmol · mg
1 · min
1.
Western blot analysis further confirmed that the decreased synthesis of
20-HETE was associated with a decrease in the levels of
CYP4A-immunoreactive proteins (Fig. 4).
Treatment with 4A2-antisense ODN did not affect renal cytochrome
P-450
(c) reductase (78 U/mg in control
vs. 76 U/mg in 4A2-AS-treated rats) or CYP2E1-dependent
p-nitrophenol hydroxylase activity
(0.35 nmol · min
1 · mg
1
in control vs. 0.32 nmol · min
1 · mg
1 in 4A2-AS-treated
rats).
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The finding that renal microvessel synthesis of 20-HETE is inhibited by
4A1-AS conflicts with reports indicating that CYP4A1 mRNA and protein
could not be detected in the renal vasculature (12). Accordingly, we
set out to investigate whether CYP4A1 is present in the renal
microvessel by performing RT-PCR with CYP4A-specific primers using
microdissected preglomerular renal microvessels freed from tubular,
connective, and parenchymal tissues. As seen in Fig.
5A,
ethidium bromide staining of the agarose gel demonstrated that all
three CYP4A isoforms (4A1, 4A2, and 4A3) were amplified from
reverse-transcribed whole kidney RNA (lanes 2, 5, and
8) as well as from their
corresponding plasmid cDNAs (lanes 3,
6, and
9). Similarly, RNA isolated from
renal microvessels revealed the presence of 317-bp and 321-bp products
for CYP4A2 and CYP4A3, respectively (Fig.
5A, lanes
4 and 7). However,
we did not detect a corresponding band (827 bp) for CYP4A1 (Fig. 5A, lane
1). Such a result could be explained by different
amplification efficiency, since the CYP4A1-specific
primers amplified a 825-bp fragment, whereas the CYP4A2- and
CYP4A3-specific primers amplified much shorter fragments.
We therefore performed additional nested PCR amplification using the
first round RT-PCR mixture as the template. As shown in
Fig. 5B (lanes
1-3), the expected 300-bp CYP4A1-specific
fragment was present in renal microvessel preparation as
well as in PCR mixtures from whole kidney and CYP4A1 plasmid. To
confirm the authenticity of these PCR products, these fragments were
transferred to nylon membranes and hybridized with each CYP4A cDNA
probe. The results of Southern blot analysis indicated that CYP4A1
along with CYP4A2 and CYP4A3 are expressed in the renal microvessels
(Fig. 5C). Western analysis using a
large gel and Sf9 recombinant CYP4A membranes as standards revealed the
presence of one major immunoreactive band with the elution profile of
the recombinant CYP4A1 and another weak immunoreactive signal having the mobility of the recombinant CYP4A2. Also, addition of CYP4A1 and
CYP4A2 recombinant proteins to the microvessel homogenates before SDS
gel separation reinforced the intensity of the signal generated by
CYP4A1 and CYP4A2 present in the renal microvessels (Fig.
6).
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Of interest, measurement of blood pressure in the in vivo experiments
revealed a significant reduction in blood pressure after treatment with
either of the antisense preparations. Thus daily injection of
4A1-antisense ODN or 4A2-antisense ODNs for 5 days reduced the blood
pressure by 17 ± 5 and 16 ± 4 mmHg, respectively (Table
1). When rats were treated with both
antisense ODNs, blood pressure was reduced by 22 ± 6 mmHg. No
significant changes were observed in rats treated with the vehicle
control or in rats treated with the scrambled ODNs (Table 1).
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DISCUSSION |
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In the present study, we evaluate the use of antisense ODNs for inhibition of specific CYP4A isoform expression and 20-HETE synthesis. One problem in assessing the specificity, potency, and efficacy of antisense ODNs against CYP-derived enzymatic activity is the lack of an in vitro system, e.g., cultured cells or cell line, expressing these activities. Many cells demonstrate a time-dependent rapid loss of CYP enzyme activity and expression following cultivation (24), which can be maintained, to some extent, with CYP inducers or cofactors (31). For example, proximal tubular epithelial cells, which are endowed with CYP enzyme activity in vivo, lose 20-HETE synthesizing capacity shortly after cultivation (18). We have used the Sf9 cells expressing CYP4A isoforms as an in vitro system to screen antisense ODNs for their specificity and efficacy in inhibiting isoform-specific expression and 20-HETE synthesis. To eliminate degradation, phosphorothioate-modified ODNs were synthesized, and to maximize uptake of ODNs, a liposomal mixture of the derivatized ODNs was administered (4, 19). Results of the in vitro studies in Sf9 cells revealed that CYP4A1- and CYP4A2/4A3-specific antisense ODNs have the ability to inhibit both protein expression and the corresponding catalytic activity, i.e., conversion of arachidonic acid to 20-HETE. These studies also yielded control ODNs that had identical base composition in a scrambled sequence but were devoid of biological activity.
The various ODNs were used for the subsequent in vivo studies where the dose, route, and duration of treatments were extrapolated in part from the in vitro experiments as well as from published studies with various antisense ODNs (4, 5, 32). We chose systemic administration, since it has been shown that phosphorothioate-modified ODNs administered intravenously accumulate mainly in the kidney and rapidly metabolize to shorter ODNs (4). Our studies clearly indicate that intravenous administration of antisense ODNs directed against CYP4A1 or CYP4A2/4A3 expression, given separately or combined, markedly inhibited 20-HETE synthesis in the renal preglomerular vessels.
The finding that the specific CYP4A1 antisense ODN preparation
inhibited renal vascular 20-HETE synthesis in vivo is intriguing. There
are several reports indicating that CYP4A1 expression in the rat male
kidney is either undetectable or very low (25, 29). Moreover, CYP4A2 is
believed to be the major CYP4A isoform expressed in the renal
microvasculature of the male rat (11, 12). The results of the in vivo
studies suggested that a significant portion of 20-HETE synthesis in
the renal vasculature is CYP4A1 derived. It is possible that despite
the clear specificity of the CYP4A1 antisense ODN in vitro (i.e., it
did not affect CYP4A2-derived 20-HETE formation), in vivo this
specificity was lost. However, RT-PCR and Western analyses provided
substantial evidence to suggest that this vascular bed expresses, in
addition to CYP4A2 and CYP4A3 mRNAs and proteins, CYP4A1 mRNA and
CYP4A1-immunoreactive protein. Recent studies in our laboratory
compared and contrasted the catalytic activities of the recombinant rat
CYP4A isoforms. These studies clearly identified CYP4A1 as the
low-Km
arachidonic acid -hydroxylase; its capacity to form 20-HETE was 20 times greater than that of CYP4A2 or CYP4A3 recombinant proteins,
namely,
Km/Vmax
of 928 vs. 72 and 22 min
1
for CYP4A1, CYP4A2, and CYP4A3, respectively (Nguyen and
Laniado-Schwartzman, unpublished data). These findings suggest that,
although present in lower amounts in the kidney, CYP4A1, with its high
arachidonic acid
-hydroxylation activity, may be a major catalyst
for the formation of 20-HETE in the rat kidney.
The in vivo studies demonstrate that in rats treated with the CYP4A1
antisense ODNs, proximal tubular synthesis was inhibited, suggesting
the significant contribution of CYP4A1 to proximal tubular 20-HETE
synthesis. In contrast, treatment with CYP4A2 antisense ODN did not
affect 20-HETE synthesis in the proximal tubules. Thus, despite the
relatively high expression of CYP4A2 in the proximal tubules (9),
CYP4A2 participation in 20-HETE production may be minimal. The
relatively low catalytic turnover of arachidonic acid -hydroxylation
by the recombinant CYP4A2 protein may explain the limited contribution
of this isoform to proximal tubular 20-HETE production. On the other
hand, one cannot exclude the possibility that the
pharmacodynamic/pharmacokinetic properties of the CYP4A2 antisense ODN
are different from the CYP4A1 antisense ODN, including differences in
binding affinity to plasma proteins and susceptibility to degradation
in the proximal tubules (4, 26). These differences may limit the
efficacy of the former ODN in its ability to inhibit 20-HETE synthesis in the proximal tubules.
The present study also demonstrates that administration of antisense ODNs against the CYP4A isoforms that catalyzed 20-HETE synthesis (CYP4A1 and CYP4A2/4A3) is associated with a significant reduction in blood pressure. Several studies have reported that 20-HETE is a constrictor of renal and extrarenal arterioles (6, 8, 11, 20). The vasoconstrictor property of 20-HETE has been attributed to its ability to potently inhibit the opening of the large-conductance Ca2+-activated K+ channel in vascular smooth muscle cells (34). Hence, the vasodepressor effect of the CYP4A antisense ODNs may be due to attenuation of a vasoconstrictor mechanism mediated by endogenous 20-HETE at renal and extrarenal sites.
In summary, this study is the first to demonstrate the use of antisense ODNs for inhibition of CYP4A isoform-specific 20-HETE synthesis. It also implicates CYP4A1 as a major 20-HETE-forming enzyme. These antisense ODNs provide the specificity needed for evaluating the contribution of each CYP4A isoform to the endogenous production of 20-HETE and thereby can be used to examine the pathophysiological role of 20-HETE.
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ACKNOWLEDGEMENTS |
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This study was supported in part by National Heart, Lung, and Blood Institute Grant PO1-HL-34300 and by Fellowship Award 970104 from the American Heart Association, New York State Affiliate.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. L. Schwartzman, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: michal_schwartzman{at}nymc.edu).
Received 16 July 1998; accepted in final form 8 October 1998.
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