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Am J Physiol Regul Integr Comp Physiol 280: R255-R261, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 1, R255-R261, January 2001

CYP4A1 antisense oligonucleotide reduces mesenteric vascular reactivity and blood pressure in SHR

Mong-Heng Wang, Fan Zhang, Jackleen Marji, Barbara A. Zand, Alberto Nasjletti, and Michal Laniado-Schwartzman

Department of Pharmacology, New York Medical College, Valhalla, New York 10595


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low Km arachidonic acid omega -hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 ± 3 to 121 ± 4 mmHg (P < 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE synthesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine (EC50 0.69 ± 0.17 vs. 1.77 ± 0.40 µM). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR.

20-hydroxyeicosatetraenoic acid; phenylephrine; myogenic response; kidney; blood pressure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENZYMES OF THE CYTOCHROME P-450 (CYP) 4A family catalyze the omega -hydroxylation of arachidonic acid and other fatty acids (2). Four isoforms have been identified in the rat: CYP4A1, CYP4A2, CYP4A3, and CYP4A8 (13, 14). Our recent studies, using baculovirus-expressed recombinant CYP4A proteins, demonstrated that CYP4A1, CYP4A2, and CYP4A3, but not CYP4A8, exhibit significant arachidonic acid omega -hydroxylating activity (18). CYP4A1 functions as an arachidonate omega -hydroxylase, with a turnover rate 20 times higher than that of CYP4A2 or CYP4A3. Furthermore, the enzyme efficiency constant (Vmax)/Km) for CYP4A1 is ~13 and 42 times higher than that of CYP4A2 and CYP4A3, respectively (18). Hence, in physiological settings, CYP4A1 expression is likely to participate prominently in determining cellular rates of synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), the product of arachidonic acid omega -hydroxylation. This notion has been substantiated in the kidney, in which we demonstrated that treatment of Sprague-Dawley rats with antisense oligodeoxynucleotides (ODNs) directed against CYP4A1 mRNA has an inhibitory influence on 20-HETE synthesis by renal vascular and tubular structures (24).

Numerous reports have implicated 20-HETE of renal origin in the regulation of vascular and tubular functions relevant to blood pressure regulation (5, 9, 20, 21, 26, 28, 29). There is evidence that 20-HETE supports the tone of renal preglomerular arterial vessels and exerts an inhibitory influence on ion transport in the thick ascending limb (3, 25). Vascular synthesis of 20-HETE also was shown to contribute to vasoconstrictor mechanisms in cerebral microvessesls (7), skeletal muscle arterioles (12), and mesenteric arteries (1). Whether or not the synthesis of 20-HETE in extrarenal vessels is dependent on CYP4A1 expression, the arachidonate omega -hydroxylase with the highest turnover rate, has not been determined as yet.

This study was designed to assess the contribution of CYP4A1 to the generation of 20-HETE by mesenteric arterial vessels and to define the functional significance of CYP4A1-catalyzed 20-HETE synthesis in relation to the regulation of vasoconstrictor responsiveness to phenylephrine and myogenic stimuli. Accordingly, mesenteric arterial vessels taken from rats treated with CYP4A1 antisense ODN or with the corresponding scrambled ODN were compared in terms of CYP4A protein expression and ability to manufacture 20-HETE. We also contrasted mesenteric arterial vessels from rats treated with CYP4A1 antisense ODN and scrambled ODN in terms of constrictor responsiveness to phenylephrine and to increases in transmural pressure. The studies were conducted in spontaneously hypertensive rats (SHR), an animal model in which the renal expression of CYP4A, in particular CYP4A1, and 20-HETE synthesis are increased (8, 16, 17, 19).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal treatment. Male SHR rats (Harlan, 6-7 wk old) were anesthetized with pentobarbital sodium (60 mg/kg ip) and instrumented with a polyethylene (PE-50) cannula into the femoral vein for ODN administration. In some experiments, rats were also instrumented with a cannula placed in the femoral artery for measurement of blood pressure. The rats were allowed 24 h to recover before treatment was commenced. Liposome-encapsulated CYP4A1 antisense ODN or scrambled ODN were injected daily as bolus at doses of 167 nmol · kg body wt-1 · day-1 for 5 days. Additional rats were treated with liposomes mixed with the ODN vehicle (5% glucose). On day 6 after the onset of treatment, the rats were killed and mesenteric arterial vessels were obtained by microdissection and used for functional studies and measurement of 20-HETE synthesis and CYP4A protein expression. In additional experiments, mesenteric arterial vessels obtained from untreated SHR were used for studying the profile of CYP4A mRNAs and for assessing vascular function.

Preparation of ODNs. The antisense ODN was targeted to bases -3 to +21 of the CYP4A1 cDNA (24), encompassing the ATG translation site codon. The base composition of the scrambled ODN was the same as that of the CYP4A1 antisense ODN, but the sequence was different. All ODN sequences were aligned to the DNA database (GenBank) using the MacVector Sequence Analysis Software. The CYP4A1 antisense ODN showed no sequence homology to CYP4A2, CYP4A3, or CYP4A8. Likewise, CYP4A1 scrambled ODN showed no sequence homology with CYP4A1 or any known CYP sequences. The phosphorothioate derivatives of the selected ODNs were synthesized and purified by Genosys Biotechnologies (Woodlands, TX). The following ODNs were used: antisense ODN for CYP4A1: 5'-CAGTGCAGAGAC- GCTCATGGT-3' (21 bases); scrambled ODN for CYP4A1: 5'-CTGACCGCAGGACTTAGATGG-3'. The potency and specificity of antisense ODNs were documented in the baculovirus/Sf9 cell system as described previously (24). Antisense and scrambled ODNs were encapsulated into a liposome mixture composed of dimethyldioctadecylammonium bromide and L-alpha -dioleylphosphatidylethanolamine (2:5 wt/wt) at a ratio of 1 µg liposomes/0.06 nmol ODN as previously described (24).

20-HETE synthesis in isolated arteries. Vessels (200-500 µm) were homogenized in a buffer containing 10 mM potassium phosphate buffer, 0.25 M sucrose, 1 mM EDTA, 0.1% Nonidet P-40 (NP-40), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and Sigma protease inhibitor cocktail (1:1,000; pH 7.4). Homogenates (50 µg protein) were incubated with arachidonic acid (20 µM) in 1 ml of assay buffer containing 100 mM potassium phosphate buffer (pH 7.4), 10 mM MgCl2, 1 mM EDTA, 1 mM NADPH, and 2 µM indomethacin at 37°C for 60 min. The reaction mixture was placed in a water bath shaker, and O2 gas was continuously blown into the incubation tubes. After incubation, [20, 20-2H2]-20-HETE (5 ng) was added as an internal standard, and the reaction mixture was acidified to pH 4 with 1 M formic acid. The mixture was extracted twice with 2 ml of ethyl acetate. The final extract was subjected to reverse-phase HPLC on a 5-m ODS-Hypersil column, 4.6 × 200 mm (Hewlett-Packard; Palo Alto, CA) using a linear gradient ranging from acetonitrile-water-acetic acid (50:50:1) to acetonitrile-acetic acid (100:0.1) at a flow rate of 1 ml/min for 30 min. Fractions coeluting with the 20-HETE standard were collected, evaporated to dryness, and derivitized to the pentafluorobenzyl bromide ester trimethylsilyl ether. Negative chemical ionization-gas chromatography/mass spectrometry (NCI-GC/MS) was performed on an HP5989A mass spectrometer (Hewlett-Packard) interfaced with a capillary gas chromatographic column (DB-1 fused silica, 10 m, 0.25-mm ID, 0.25-µm film thickness, J&W Scientific; Rancho Cordova, CA) and programmed from 180-300°C at 25°C/min using helium as the carrier gas. Single ions were monitored with m/z 391 corresponding to the derivatized 20-HETE and m/z 393 for the derivatized [20,20-2H2]-20-HETE internal standard. Total 20-HETE in each sample was determined by comparison of the ratio of ion intensities (391:393) vs. a standard curve of derivatized 20-HETE/[20,20-2H2]-20-HETE molar ratio obtained from NCI-GC/MS analysis.

Western blot analysis. Mesenteric arteries (200-500 µm) were homogenized in a buffer containing 0.1 M potassium phosphate buffer, 0.25 M sucrose, 1 mM EDTA, 0.1% NP-40, 0.1 mM PMSF, and Sigma protease inhibitor cocktail (1:1,000; pH 7.4). Proteins were separated by electrophoresis on an 8% SDS-polyacrylamide gel at 120 V for 2 h. The proteins were transferred electrophoretically to a polyvinylidene fluoride membrane in a transfer buffer consisting of 25 mM Tris · HCl, 192 mM glycine, and 20% methanol (vol/vol). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 10 mM Tris, 0.1% Tween 20, and 150 mM NaCl for 1.5 h and then washed three times with TBS. The membranes were incubated for 1 h with goat anti-rat CYP4A1 polyclonal antibody (1:1,000; Gentest; Woburn, MA) at room temperature, washed with TBS solution, and further incubated with 1:5,000 dilution of alkaline phosphatase-conjugated second antibody (Sigma; St. Louis, MO) for 1 h. After being washed with TBS three times, the membranes were blotted dry and incubated with 1 ml of Vistra enhanced chemical fluorescence substrate (Amersham). The membranes were air-dried completely, and immunoreactive proteins were detected by the Storm PhosphoImager system (Molecular Dynamics; Sunnyvale, CA) and quantified by ImageQuant analysis.

RT-PCR. Microdissected mesenteric arterial vessels (200-250 µm) were placed in 100 µl of phenol and guanidinium thiocyanate solution (TRIzol, GIBCO; Life Technologies). RNA was extracted by the single-step guanidinium thiocyanate-phenol-chloroform method and resuspended in 5 µl of nuclease-free water. A reverse-transcription reaction was performed using a First-strand cDNA synthesis kit (Pharmacia Biotech; Milwaukee, WI). Briefly, 5 µl of RNA (40 ng) were added to 15-µl reverse-transcription reaction mixture containing 45 mM Tris (pH 8.3), 68 mM KCl, 15 mM 1,4-dithiothreitol, 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 RT. 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 the first strand reaction. PCR was also performed using the CYP4A plasmids (10 pg) as templates 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 35 times through a 1-min denaturing step at 95°C, a 1-min annealing step at 60°C, and a 1-min extension step at 72°C. After the cycling procedure, a final 10-min elongation step at 72°C was performed. The specific CYP4A1, CYP4A2, and CYP4A3 primers were designed to amplify 351-, 317-, and 321-bp fragments from each of the correponding cDNAs. The sequences of the primers used were as follows: CYP4A1: 5'-CTCTTACTTGCCAGAATGGAGAA-3' (forward primer), 5'-GACTTGGATACCCTTGGGTAAAG-3' (backward primer); CYP4A2: 5'-AGATCCAAA- GCCTTATCAATC-3' (forward primer), 5'-CAGCCTTGGTGTAGGACC-T-3' (backward primer); CYP4A3: 5'-CAAAGGCTTCTGGAATTTATC-3' (forward primer), 5'-CAGCCTTGGTGTAGGACCT-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 (N-hybond, 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).

Measurement of isometric tension in vascular rings. Mesenteric arteries (200-250 µm ID) were cut into ring segments ~2 mm in length. Vascular rings were mounted on wires in the chambers of a multivessel myograph (J. P. Trading; Aarthus, Denmark) filled with Krebs buffer (37°C). The buffer was gassed with 95% O2-5% CO2. After equilibration for 30-60 min, the internal circumference of the vessels was set at a value equivalent to 90% of what they would have been in vitro when relaxed under a transmural pressure of 80 mmHg. Isometric tension was monitored continuously before and after each experimental intervention and is expressed as milliNewtons per millimeter of vessel length. Experiments were initiated after a 30- to 60-min stabilization interval. The constrictor response to 60 mM KCl was determined in each vessels. Subsequently, after washing, a cumulative concentration-response curve to phenylephrine (0.1-50 µmol/l) was constructed.

Evaluation of pressure-diameter relationships in mesenteric arterioles. Mesenteric arterioles (70 µm ID; 1-2 mm in length) were transferred to a water-jacketed vessel chamber (1 ml volume) containing Krebs buffer at room temperature. The vessels were mounted on a proximal micropipette connected to a pressure servo controller. Subsequently, the lumen of the vessel was flushed to remove residual blood, and the end of the vessel was mounted on a micropipette connected to a three-way stopcock. After the stopcock was closed, the intraluminal pressure was allowed to increase slowly until it reached 80 mmHg. The vessels were superfused with Krebs bicarbonate buffer (1 ml/min) at 37°C gassed with 95% O2 and 5% CO2 for a 60-min period of equilibration at 80 mmHg. The vessel chamber was mounted on the stage of a microscope fitted with a video camera leading to a video caliper, monitor, and recorder. Only preparations that developed spontaneous tone during equilibration were used. The pressure-diameter relationship was studied as described previously (15). The intraluminal pressure was decreased to ~0 mmHg, and after 10 min, it was increased in 20-mmHg steps until it reached 100 mmHg. The pressure was maintained for 5-10 min at each pressure step to allow time to reach a steady-state diameter. Before concluding an experiment, the superfusion buffer was changed to calcium-free Krebs bicarbonate buffer containing 1 mM EGTA and the pressure-diameter relationship was examined again to obtain the passive diameter of the vessel at each level of intraluminal pressure. Vascular diameters at each pressure level were expressed as a percentage of passive diameter (15).

Statistical analysis. Data are expressed as means ± SE. Concentration-response data derived from each vessel were fitted separately to a logistic function by nonlinear regression, and EC50 was calculated using commercially available software (Prism 2.01, GraphPAD software, San Diego, CA). Concentration-response data were analyzed by a two-way analysis of variance followed by Duncan's multiple-range test. Other data were analyzed by Student's t-test for paired or unpaired observations as appropriate. The null hypothesis was rejected at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The expression of CYP4A mRNAs was examined by RT-PCR with CYP4A-specific primers in mesenteric arteries. RNA isolated from mesenteric arteries revealed the presence of the expected PCR products of 351, 317, and 321 bp for CYP4A1, CYP4A2, and CYP4A3, respectively (Fig. 1A). The specificity of these primers was examined using CYP4A1, CYP4A2, and CYP4A3 cDNAs as templates for the PCR reactions (Fig. 1B).


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  		Fig. 1.   RT-PCR of mesenteric arteries with cytochrome P-450 4A (CYP4A)-isoform specific primers. A: mesenteric arterial total RNA (40 ng) was extracted and amplified with CYP4A isoform-specific primers. RT-PCR products were separated on agarose gel and stained with ethidium bromide. Lane 1: CYP4A1 specific primers (351 bp PCR product); lane 2: CYP4A2 specific primers (317 bp PCR product); lane 3: CYP4A3 specific primers (321 bp PCR product); M, size markers. B: full-length CYP4A1, CYP4A2, and CYP4A3 cDNA plasmids (10 ng) were amplified by PCR for 25 cycles with each of the CYP4A primer pairs. Lanes 1, 4, and 7: CYP4A1 specific primers (351 bp PCR product); lanes 2, 5, and 8: CYP4A2 specific primers (317 bp PCR product); lanes 3, 6, and 9: CYP4A3 specific primers (321 bp PCR product).

As seen in Fig. 2, Western blot analysis in homogenates of mesenteric arterial vessels revealed the presence of CYP4A immunoreactive proteins. The levels of CYP4A immunoreactive proteins in specimens taken from rats treated with CYP4A1 antisense ODN (1.99 ± 0.23 arbitrary density units; n = 3) was greatly reduced (P < 0.05) relative to the levels in specimens taken from rats treated with the scrambled ODN (7.52 ± 0.40 arbitrary density units; n = 3). Figure 2 also shows that mesenteric arterial vessels manufacture 20-HETE. The rate of 20-HETE synthesis in vessels taken from rats treated with CYP4A1 antisense ODN was greatly decreased (P < 0.05) relative to the rate in vessels taken from rats treated with scrambled ODN.


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  		Fig. 2.   CYP4A expression and 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis in mesenteric arteries. Top: representative immunoblots of homogenates prepared from mesenteric arteries taken from scrambled oligonucleotide (ODN)-treated rats (4A1-S) and CYP4A1 antisense-treated rats (4A1-AS). Bottom: 20-HETE synthesis in mesenteric arteries from scrambled ODN-treated rats (4A1-S) and CYP4A1 antisense-treated rats (4A1-AS). Results are the means ± SE (n = 3). * P < 0.05 from 4A1-S treatment.

Treatment of SHR with CYP4A1 antisense ODN impacted significantly on the level of mean arterial pressure and mesenteric vascular function. Daily injections of CYP4A1 antisense ODN for 5 days significantly reduced mean arterial blood pressure in SHR from 137 ± 3 to 110 ± 6 and 121 ± 4 mmHg after 3 and 5 days of treatment, respectively (Table 1). On the other hand, when rats were treated with CYP4A1 scrambled ODN, blood pressure was not affected significantly (Table 1).

                              
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  		Table 1.   Effect of antisense ODNs on mean arterial blood pressure in SHR

Shown in Fig. 3, phenylephrine elicited a constriction-dependent increase of isometric tension in rings of small mesenteric artery. At maximally effective concentrations of phenylephrine, the constrictor effect was comparable in vessels taken from rats treated with CYP4A1 antisense ODN, scrambled ODN, and from control untreated rats. However, at submaximally effective concentrations of phenylephrine, constrictor responses in vessels taken from rats treated with CYP4A1 antisense ODN were surpassed by the responses in vessels taken from either rats treated with scrambled ODN or from control untreated rats. Thus the concentration-response curve to phenylephrine is shifted to the right in small mesenteric arteries of rats treated with CYP4A1 antisense ODN, reflecting an increase in EC50 from 0.69 ± 0.17 µM in vessels from rats treated with scrambled ODN and 0.65 ± 0.15 µM in vessels from untreated rats to 1.77 ± 0.40 µM in vessels from rats treated with CYP4A1 antisense ODN. The inhibitory effect of CYP4A1 antisense ODN treatment on constrictor responses to phenylephrine in small mesenteric arteries was offset, in part, by the inclusion of exogenous 20-HETE (1 µmol/l) in the bathing media, causing the EC50 for phenylephrine to decrease to 1.11 ± 0.23 µM.


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  		Fig. 3.   Concentration-response curves to phenylephrine in small mesenteric arteries taken from control untreated spontaneously hypertensive rats (SHR; untreated) and from SHR treated with 4A1-AS or 4A1-S ODNs (167 nmol · kg body wt-1 · day-1 for 5 days). Data are expressed as means ± SE (n = 10); *P < 0.05 relative to 4A1-S treatment.

The results of experiments comparing mesenteric arterioles from untreated rats, rats treated with CYP4A1 antisense ODN, and rats treated with scrambled ODN in terms of pressure-diameter relationship are depicted in Fig. 4. Stepwise elevation of intramural pressure over the range of 20-100 mmHg elicited pressure-related reduction of arteriolar diameter expressed as a percentage of the passive diameter in the absence of extracellular calcium. The pressure-induced constriction of mesenteric arterioles over the range of 60-100 mmHg was less intense in vessels obtained from rats treated with CYP4A1 antisense ODN than in vessels taken from either rats treated with scrambled ODN or untreated rats. The inhibitory effect of CYP4A1 antisense ODN treatment on pressure-induced vasoconstriction was offset by the inclusion of 20-HETE (1 µmol/l) in the superfusion buffer.


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  		Fig. 4.   Pressure-diameter relationship in mesenteric arteries isolated from control untreated SHR (untreated) and from SHR treated with antisense (4A1-AS) or scrambled (4A1-S) ODNs (167 nmol · kg body wt-1 · day-1 for 5 days). Data are expressed as means ± SE (n = 6); *P < 0.05 relative to 4A1-S treatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study in SHR demonstrates that mesenteric arterial vessels manufacture 20-HETE and express CYP4A immunoreactive proteins along with mRNA coding for CYP4A1, CYP4A2, and CYP4A3. These CYP4A isoforms catalyze omega -hydroxylation of arachidonic acid to 20-HETE. However, the enzyme efficiency constant of the recombinant CYP4A1 is much higher than that of CYP4A2 or CYP4A3 (18), suggesting that it may be the principal contributor of endogenous 20-HETE formation. To assess the contribution of CYP4A1 to 20-HETE formation in arterial vessels, we used specific antisense ODN for targeting CYP4A1 expression. This approach is based on reports that antisense ODN binds to its target mRNA bringing about inhibition of gene product expression. We showed that the mesenteric, similar to the renal vessels (24), express mRNAs for CYP4A1 as well as for CYP4A2 and CYP4A3. Moreover, we found that 20-HETE synthesis and CYP4A protein expression in arterial vessels from rats treated with CYP4A1 antisense ODN are reduced relative to corresponding data in rats treated with scrambled ODN or untreated rats. These findings support the conclusion that CYP4A1 is one of the CYP4A isoforms that contribute importantly to the generation of 20-HETE in the mesenteric vasculature.

Several studies have shown that 20-HETE stimulates contraction of vascular smooth muscle. In large arterial vessels, the contracting action of 20-HETE is mediated by a product of cyclooxygenase, presumably a 20-hydroxy-prostaglandin endoperoxide metabolite (4). In small arterial vessels of the kidney and brain, 20-HETE elicits vascular contraction via a cyclooxygenase-independent mechanism that involves inhibition of the large-conductance Ca2+-activated K+ channel leading to vascular smooth muscle cell depolarization and elevation in cytosolic Ca2+ (6, 10, 23, 27). In the renal circulation, endogenous 20-HETE has been implicated in the mediation of vasconstriction associated with activation of tubuloglomerular feedback and renal blood flow autoregulation (28, 29). 20-HETE also plays a central role in the implementation of myogenic constrictor responses to elevations of transmural pressure in small renal and cerebral arterial vessels (7, 10, 11). The current study provides evidence that 20-HETE of vascular origin contributes importantly to the implementation of vasoconstrictor mechanisms in the mesenteric circulation. The diminished EC50 for phenylephrine-induced contraction of small mesenteric arteries taken from rats treated with CYP4A1 antisense ODN suggests that vascular sensitivity to the constrictor agonist is subjected to stimulation by endogenous 20-HETE. Similarly, the observation that the constrictor response elicited by increases of intraluminal pressure in mesenteric arteries is enhanced in vessels taken from scrambled ODN-treated rats or from untreated rats suggests that 20-HETE amplifies myogenic behavior in the mesenteric circulation. The conclusion that endogenous 20-HETE sensitizes vascular smooth muscle to constrictor stimuli is in agreement with a recent report that pharmacological inhibition of 20-HETE synthesis attenuates angiotensin II-induced mesenteric vasoconstriction in vivo (1). The facilitatory actions of endogenous 20-HETE on vascular responsiveness both to a constrictor agonist and myogenic stimuli may result from the ability of this eicosanoid to decrease the activity of large Ca2+-activated K+ channels (23, 27).

In the present study, treatment of SHR with CYP4A1 antisense ODN reduced blood pressure significantly, whereas treatment with scrambled ODN did not. Likewise, a previous report showed that the blood pressure of Sprague-Dawley normotensive rats also falls with the administration of CYP4A1 antisense ODN (24). One interpretation of these observations is that, at vascular sites, CYP4A1-derived 20-HETE supports blood pressure in both normotensive and hypertensive settings, presumably by magnifying vasoconstrictor responsiveness in both renal and extrarenal vascular beds (5). But there is evidence that 20-HETE produced in the thick ascending limb of the loop of Henle inhibits ion transport in this segment of the nephron (3, 25), promoting salt-water excretion that is conducive to lowering blood pressure (22). This point merits consideration in relation to the present study, because the blood pressure reduction caused by treatment of SHR with CYP4A1 antisense ODN tended to become smaller with time. Thus it is conceivable that the vasodepressor response resulting from diminished vascular production of 20-HETE in SHR treated with CYP4A1 antisense ODN is, in part, masked by the salt-water retention expected to result from diminished 20-HETE synthesis at renal tubular sites. Our current study offers no information on the effect of CYP4A1 antisense ODN on the pressure-natriuresis relationship of SHR as well as on the blood pressure of an appropriate strain of control normotensive rats. Therefore, the precise contribution of CYP4A1-derived 20-HETE to setting the level of blood pressure in SHR and normotensive rats is yet to be determined.

In summary, this study demonstrates that treatment of SHR with CYP4A1 antisense ODN reduces 20-HETE synthesis and CYP4A immunoreactive proteins in mesenteric arterial vessels. It also documents that treatment with CYP4A1 antisense ODN decreases the sensitivity of small mesenteric arteries to phenylephrine-induced vascular contraction and the intensity of myogenic constrictor responses to elevation in intraluminal pressure in mesenteric arterioles. These data suggest that CYP4A1 contributes prominently to the generation of 20-HETE in mesenteric arterial vessels and that CYP4A1-derived 20-HETE enhances vascular sensitivity to phenylephrine and responsiveness to myogenic stimuli in the mesenteric vasculature.

Prespective

The results of the present study in mesenteric arterial vessels of SHR foster the notion that CYP4A1-catalyzed synthesis of 20-HETE sensitizes vascular smooth muscle both to myogenic and hormonal stimuli. Hence, 20-HETE of vascular origin may contribute to vascular tone by amplifying the response of the mesenteric vasculature to known neurohormonal vasoconstrictor systems. Such a regulatory action of vascular 20-HETE may impact on total peripheral vascular resistance, because 20-HETE synthesis also has been documented in other vascular beds including kidney, skeletal muscle, and brain. If so, the reduction of arterial pressure brought about by treatment of SHR with CYP4A1 antisense ODN may be explained by reduction of total peripheral resistance consequent to a generalized decrease in vascular 20-HETE synthesis. Although our study fosters the notion that CYP4A1-catalyzed synthesis of 20-HETE supports blood pressure in the SHR, a complete understanding of the role of this eicosanoid in long-term regulation of blood pressure necessitates additional information on the integrated impact of the renal tubular actions of endogenous 20-HETE that promote natriuresis and the vascular actions that promote vasoconstriction.


    ACKNOWLEDGEMENTS

This study was supported, in part, by a grant from the National Heart, Lung, and Blood Institute (PO1-34300) and by American Heart Association grants to M.-H. Wang (99-30277T) and F. Zhang (99-30291T).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Laniado-Schwartzman, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: michal_schwartzman{at}nymc.edu).

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. Section 1734 solely to indicate this fact.

Received 13 March 2000; accepted in final form 18 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 280(1):R255-R261
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