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Am J Physiol Heart Circ Physiol 288: H1557-H1565, 2005. First published December 2, 2004; doi:10.1152/ajpheart.00755.2004
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Effects of high-salt diet on CYP450-4A {omega}-hydroxylase expression and active tone in mesenteric resistance arteries

Jingli Wang, Richard J. Roman, John R. Falck, Lourdes de la Cruz, and Julian H. Lombard

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 27 July 2004 ; accepted in final form 29 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study investigated the role of changes in the expression of the cytochrome P-450 4A (CYP450-4A) enzymes that produce 20-hydroxyeicosatetraenoic acid (20-HETE) in modulating the responses of rat mesenteric resistance arteries to norepinephrine (NE) and reduced PO2 after short-term (3-day) changes in dietary salt intake. The CYP450-4A2, -4A3, and -4A8 isoforms were all detected by RT-PCR in arteries obtained from rats fed a high-salt (HS, 4% NaCl) diet, whereas only the CYP450-4A3 isoform was detected in vessels from rats fed a low-salt (LS, 0.4% NaCl) diet. Expression of the 51-kDa CYP450-4A protein was significantly increased by a HS diet. Inhibiting 20-HETE synthesis with 30 µM N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) reduced the vasoconstrictor response to NE in arteries obtained from rats fed either a LS or HS diet, but NE sensitivity after DDMS treatment was significantly lower in vessels from rats on a HS diet. DDMS treatment also restored the vasodilator response to reduced PO2 that was impaired in arteries from rats on a HS diet. These findings suggest that 1) a HS diet increases the expression of CYP450-4A enzymes in the mesenteric vasculature, 2) 20-HETE contributes to the vasoconstrictor response to NE in mesenteric resistance arteries, 3) the contribution of 20-HETE to the vasoconstrictor response to NE is greater in rats fed a HS diet than in rats fed a LS diet, and 4) upregulation of the production of 20-HETE contributes to the impaired dilation of mesenteric resistance arteries in response to hypoxia in rats fed a HS diet.

cytochrome P-450; isoform; hypertension; vascular bed; hydroxyeicosatetraenoic acid

THE VASOCONSTRICTOR 20-HYDROXYEICOSATETRAENOIC acid (20-HETE), which is a metabolite of arachidonic acid, is formed in a number of vascular beds secondary to the action of cytochrome P-450 4A (CYP450-4A) {omega}-hydroxylase. It appears that 20-HETE plays an important role in regulating vascular tone and reactivity in renal (18, 28, 47), cerebral (13–15, 35), and skeletal muscle (16, 21, 22) arterioles and mesenteric arteries (3, 40, 45). For example, previous studies have demonstrated that a high-salt (HS) diet increases the sensitivity of cremaster muscle arterioles to the vasoconstrictor effects of ANG II, and that inhibitors of the formation of 20-HETE shift the concentration-response curve for ANG II to the right (8). ANG II increases the production of 20-HETE by renal preglomerular arterioles (4), and inhibiting 20-HETE formation attenuates the pressor response to intravenous infusion of ANG II in vivo (1). Other studies suggest that 20-HETE contributes to phenylephrine-induced constriction of mesenteric resistance arteries (45) and to O2-induced constriction of skeletal muscle arterioles in rats (21) and hamsters (26). Reductions in vascular 20-HETE levels also may contribute to hypoxic dilation of skeletal muscle resistance arteries (10). Taken together, these observations suggest that 20-HETE plays an important role in mediating the response of arterioles to vasoactive stimuli.

Several isoforms of the CYP450-4A family are expressed in blood vessels from different vascular beds of rats (13, 14, 29, 33, 37). However, there is considerable heterogeneity in the isoforms that are expressed in vessels obtained from different vascular beds (33), and the role of 20-HETE in regulating the responses of resistance arteries to vasoconstrictor stimuli remains to be established. A recent study by our laboratory (37) demonstrated that CYP450-4A3 and -4A8 are the major isoforms of CYP450-4A {omega}-hydroxylase expressed in arterioles microdissected from cremaster muscles of Sprague-Dawley rats, whereas another study (39) demonstrated that the CYP450-4A1, -4A2, and -4A3 isoforms are all found in preglomerular arterioles isolated from kidneys of Sprague-Dawley rats. Although the CYP450-4A2 isoform seems to be the major isoform in renal microvessels (18, 19), Wang et al. (39) suggested that the production of 20-HETE by renal microvessels may be primarily determined by CYP450-4A1, because the catalytic activity of the CYP450-4A1 isoform is 40 times higher than that of the CYP450-4A2 and -4A3 isoforms (29).

Recent studies have suggested that the CYP450-4A1 isoform is expressed in the mesenteric vascular bed of spontaneously hypertensive rats (40). However, it remains to be determined whether changes in dietary salt intake alone (without hypertension) alter the expression of CYP450-4A isoforms in resistance arteries, and whether changes in the expression of CYP450-4A isoforms can affect the reactivity of these vessels to vasoactive stimuli. To test this hypothesis, the present study used RT-PCR to identify the isoforms of CYP450-4A mRNA expressed in mesenteric resistance arteries of Sprague-Dawley rats fed a low-salt (LS) or HS diet for 3 days. Western blots were used to determine the effects of changes in salt intake on the expression of CYP450-4A protein in these vessels. Finally, we compared the responses to norepinephrine (NE) and reduced PO2 in mesenteric resistance arteries from rats fed a LS or HS diet before and after blockade of the formation of 20-HETE by treating the vessels with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animal preparation. Experiments were performed on male Sprague-Dawley rats that weighed 250–305 g (8–10 wk old) from Harlan Laboratories (Indianapolis, IN). The rats were housed in the Animal Resource Center at the Medical College of Wisconsin, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, and all experimental protocols were approved by the Medical College of Wisconsin Animal Care Committee. The animals were fed standard rat diet with tap water ad libitum. Three days before the experiment, the standard diet was replaced by a LS (0.4% NaCl) or HS (4% NaCl) diet purchased from Dyets (Bethlehem, PA).

General procedures. The rats were anesthetized with pentobarbital sodium (60 mg/kg ip) and positioned on a thermostatically regulated heating pad. The carotid artery was cannulated and arterial blood pressure was measured directly. Mesenteric resistance arteries were carefully isolated and placed in either cold (4°C) physiological salt solution (PSS) or cold (4°C) RNAlater solution (Ambion; Austin, TX) depending upon the specific protocol. The PSS was bubbled with a 21% O2-5% CO2-74% N2 gas mixture and had the following composition (in mM): 119 NaCl, 4.7 KCl, 1.17 MgSO4, 1.6 CaCl2, 1.18 NaH2PO4, 24 NaHCO3, 0.026 EDTA, and 5.5 glucose.

Evaluation of vascular reactivity to NE and reduced PO2. Studies of isolated cannulated resistance arteries were conducted using standard procedures described previously (6, 7, 24, 27). After isolation, mesenteric arteries (100–300 µm internal diameter) were transferred to a heated (37°C) chamber and cannulated with tapered glass micropipettes. The inflow pipette was connected to a reservoir system that allowed the intraluminal pressure and gas concentrations of the luminal perfusate to be controlled. The artery was secured with 10-0 nylon suture (22 µm diameter; Look; Norwell, MA), and any side branches were tied off with a single strand teased from 2-0 silk suture (Ethicon; Somerville, NJ). The artery was then stretched to approximate its in situ length, and intraluminal pressure was set at 60 mmHg. After mounting was completed, the artery was allowed to equilibrate for 30 min with continuous superfusion and perfusion of the lumen with PSS equilibrated with 21% O2. Vessel diameters were measured by video microscopy as previously described (6, 27).

The internal diameter of the vessel was measured during a control period and after various concentrations of NE (10 nM to 10 µM) were added to the bath. In another series of experiments, arteries were preconstricted with 1 µM NE and equilibrated with 21% O2 before the control diameter of the vessels was measured. The O2 concentration of the PSS in the tissue bath (superfusate) and the inflow reservoir (luminal perfusate) were then reduced by equilibrating the PSS with a 0% O2-5% CO2-95% N2 gas mixture. This procedure results in a reduction of both perfusate PO2 and superfusate PO2 from the control value of ~140 to ~40–45 mmHg during equilibration of the reservoirs with the 0% O2 gas mixture (6).

To determine the contribution of 20-HETE to NE-induced vasoconstriction and hypoxic dilation in the vessels, the formation of the compound was inhibited with DDMS, which is a specific inhibitor of 20-HETE synthesis by CYP450-4A {omega}-hydroxylase (33, 38). In these experiments, the responses to increasing concentrations of NE or to a simultaneous reduction of perfusate and superfusate O2 concentrations from 21 to 0% O2 were determined during an initial control period and were then retested in the presence of DDMS. In the NE experiments, a separate series of time-control experiments was performed by repeating the NE concentration-response curve in the absence of DDMS.

RT-PCR. RNA was extracted using the single-step guanidinium thiocyanate-phenol-chloroform method (19). Briefly, the isolated tissue was placed in an Eppendorf tube that contained 1 ml of TRIzol reagent (Invitrogen Life Technologies; Carlsbad, CA) and homogenized on ice. After homogenization, 20 µl of proteinase K (10 mg/ml; Sigma Chemical; St. Louis, MO) was added, and the homogenate was incubated at 55°C for 10 min. The homogenate was incubated at room temperature for an additional 15 min, 200 µl of chloroform (Fisher Scientific; Fairlawn, NJ) was added, and the sample was then incubated at room temperature for 3 min. After centrifugation at 12,000 g for 15 min at 4°C, the upper aqueous phase that contained the RNA was transferred to a new Eppendorf tube, and RNA was precipitated using 500 µl of absolute isopropyl alcohol (Sigma Chemical). The RNA pellet was washed with 1 ml of 75% ethanol and was repelleted.

To eliminate any potential genomic DNA contamination, the RNA pellet was dissolved in 50–100 µl of nuclease-free water and incubated with 1 µl of RQ1 RNase-free DNase (Promega; Madison, WI) at 37°C for 1 h. The reaction was stopped by addition of RQ1 DNase stop solution (Promega) and incubation at 65°C for 10 min. After treatment with DNase, the RNA was precipitated with isopropanol. The final RNA pellet was reconstituted in 30–50 µl of nuclease-free water, and the concentration of RNA was determined by measuring the absorbance at a wavelength of 260 nm in a spectrophotometer. RT reactions were performed using the Omniscript RT Kit (Qiagen; Hilden, Germany) and incubated at 37°C for 120 min, which was followed by inactivation at 93°C for 5 min and rapid cooling on ice.

The PCR was performed using the HotStarTaq Master Mix Kit (Qiagen) and was conducted in the presence of 1.25 µl (0.5 µM) of each corresponding primer (CYP450-4A1, -4A2, -4A3, and -4A8), 4 µl of the RT or no-RT product, variable amounts of distilled water, and 25 µl of HotStarTaq Master Mix. The total volume of the final reaction mixture was 50 µl. The present studies employed specific oligonucleotide primers developed for the CYP450-4A1, -4A2, and -4A3 isoforms by Ito et al. (19) and for the CYP450-4A8 isoform by Gebremedhin et al. (13). The primers used in the PCR studies were purchased from Operon Technologies (Alameda, CA) and had the following sequences: CYP450-4A1 forward, 5'-GTA TCC AAG TCA CAC TCT CCA-3'; CYP450-4A1 reverse, 5'-CAG GAC ACT GGA CAC TTT ATT G-3'; CYP450-4A2 forward, 5'-AGA TCC AAA GCC TTA TCA ATC-3'; CYP450-4A2 reverse, 5'-CAG CCT TGG TGT AGG ACC T-3'; CYP450-4A3 forward, 5'-CAA AGG CTT CTG GAA TTT ATC-3'; CYP450-4A3 reverse, 5'-CAG CCT TGG TGT AGG ACC T-3'; CYP450-4A8 forward, 5'-ATC CAG AGG TGT TTG ACC CTT AT-3'; CYP450-4A8 reverse, 5'-AAT GAG ATG TGA GCA GAT GGA GT-3'; GAPDH forward, 5'-CAC GGC AAG TTC AAT GGC ACA-3'; GAPDH reverse, 5'-GAA TTG TGA GGG AGA GTG CTC-3'. The CYP450-4A1, -4A2, -4A3, -4A8, and GAPDH primer pairs amplify fragments of 902, 321, 321, 349, and 970 bp, respectively.

Liver RNA was used as a positive control. Negative controls included experiments in which distilled water rather than c-DNA was added to the PCR reactions to exclude nonspecific contamination, or the reverse transcriptase step was omitted before PCR to exclude any potential genomic DNA contamination. To check the quality of the extracted RNA sample and PCR reaction, forward and reverse primers for the housekeeping gene GAPDH (Operon Technologies) and the G3PDH control reagent (Clontech Laboratories; Palo Alto, CA) were used for each PCR reaction.

In these experiments, the PCR program started with an initial heat-activation step at 95°C for 15 min. Subsequently, the reaction was cycled 40 times between 94°C (denaturation) for 1 min, 56°C (annealing) for 2 min, and 72°C (extension) for 1 min. All samples were incubated at 72°C for an additional 10 min after the completion of the final cycle. Aliquots (10 µl) of each PCR reaction product were separated on a 1% agarose gel that contained ethidium bromide (1 µg/ml) at 90 V for 45 min and were visualized under UV light. The molecular weight of the PCR product was determined by comparison with a 100-bp DNA ladder.

Western blots. For Western blotting, isolated mesenteric resistance arteries were homogenized in 100 µl of a solution that contained 250 mM sucrose, 1 mM EDTA, and 0.4 µl of protease inhibitor cocktail (P-8340; Sigma Chemical) in a 10 mM potassium phosphate buffer (pH 7.7). Tissue debris and nuclear fragments were removed by centrifugation at 410 and 1,230 g for 15 min at 4°C. The amount of dissolved protein in the supernatant was determined using the Bradford assay (Bio-Rad Laboratories) with bovine serum albumin as a standard.

Protein from the vessel homogenates (50 µg of protein for the 50-kDa band and 10 µg of protein for the 85-kDa glycosylated band) were separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked overnight in Tris-buffered saline (10 mM Tris and 150 mM NaCl) that contained 7.5% nonfat dry milk. The following day, the membrane was washed with Tris-buffered saline solution that contained 0.1% Tween 20 (TBST) and was incubated for 2 h with a 1:2,000 dilution of goat CYP450-4A1 antibody (catalog no. 299230; BD Biosciences; Woburn, MA) that cross-reacts with all of the CYP450-4A isoforms. After the membrane was washed with the TBST, it was incubated in a 1:5,000 dilution of horseradish peroxidase-coupled donkey anti-goat secondary antibody (Santa Cruz Biotechnology; Santa Cruz, CA). Antigen-antibody reactions were detected using SuperSignal substrate (Pierce; Rockford, IL) exposed to Kodak Biomax ML film and were developed in a Konica SRX-101 developer.

Samples from animals fed LS and HS diets were run on the same gel to avoid differences arising from gel-to-gel variation. Densitometry values (in pixels) were obtained using UnScanIT 5.1 software. The expression of CYP450-4A bands for each animal was measured as a percentage of the density of 0.1 µg of the protein standard furnished by the supplier (hepatic microsomal CYP450-4A1 protein from clofibrate-treated male rats), which was run on each gel.

Statistical analysis. In the present study, statistical significance of nonparametric variables, i.e., expression of individual CYP450-4A isoforms, was determined using a z test with the Yates' correction applied to the calculation (23). The latter test determines whether a specific isoform exhibits a statistically significant expression within a given tissue. For comparisons between two groups, raw data were summarized as means ± SE, and differences were evaluated using an unpaired Student's t-test. In the experiments that evaluated vasoconstrictor responses to NE, differences within or between groups were assessed by repeated two-way ANOVA with a subsequent post hoc Tukey's test. EC50 was calculated using GraphPad Prism 4 software, and differences in EC50 within groups were evaluated using a paired Student's t-test. Statistical significance was taken as P < 0.05.


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Effects of HS diet on arterial blood pressure. Blood pressure was not affected by a short-term HS diet. Mean arterial pressure as measured by direct arterial cannulation averaged 114 ± 2.9 mmHg (n = 12) in rats fed a LS diet and 115 ± 3.5 mmHg (n = 11) in rats fed a HS diet.

Effects of HS diet on expression of mRNA for CYP450-4A isoforms. The results of RT-PCR studies to evaluate expression of mRNA for specific CYP450-4A isoforms are depicted in Fig. 1 and summarized in Table 1. In these experiments, only CYP450-4A3 mRNA was detected by RT-PCR in mesenteric arteries of Sprague-Dawley rats fed a LS diet. In contrast, arteries obtained from rats fed a HS diet expressed mRNA for the CYP450-4A2, -4A3, and -4A8 isoforms. All four CYP450-4A isoforms (-4A1, -4A2, -4A3, and -4A8) were detected in every sample from liver.



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  		Fig. 1. Representative gels from RT-PCR studies showing the expression of mRNA for various isoforms of cytochrome P-450 4A (CYP450-4A) {omega}-hydroxylase in liver (left) and isolated mesenteric resistance arteries (middle) of normotensive Sprague-Dawley rats on low-salt (LS, top) and high-salt (HS, bottom) diet for 3 days. L, MA, and P represent GAPDH reaction in liver, mesenteric arteries, and G3PDH control reagent, respectively. H2O represents distilled water control for nonspecific contamination.

 

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  		Table 1. Expression of cytochrome P-450 4A isoforms in isolated mesenteric arteries and livers of normotensive Sprague-Dawley rats on LS and HS diets

 
Expression of CYP450-4A {omega}-hydroxylase protein in mesenteric arteries of rats fed LS or HS diet. Western blotting was performed in mesenteric arteries from rats fed a LS or HS diet to confirm that CYP450-4A is expressed at the protein level in these vessels (Fig. 2). In these experiments, expression of the 51-kDa band, which corresponds to the microsomal CYP450-4A protein standard, was significantly elevated in vessels obtained from rats fed a HS diet compared with rats fed a LS diet (Fig. 2C). Expression of an immunoreactive 85-kDa band, which corresponds to the glycosylated form of the enzyme (21), was not significantly different in vessels from animals on LS and HS diets (Fig. 2D).



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  		Fig. 2. Western blots show expression of 85- and 51-kDa CYP450-4A immunoreactive protein in isolated mesenteric arteries of Sprague-Dawley rats on LS and HS diet for 3 days. A: lane 1, 0.01 µg of commercial CYP450-4A1 microsomal protein standard from clofibrate-treated male rat liver; lane 2, 10 µg of protein from homogenates of isolated mesenteric arteries from rats on LS diet; and lane 3, 10 µg of protein from homogenates of isolated mesenteric arteries from rats on a HS diet. B: lane 1, 0.01 µg of commercial CYP450-4A1 microsomal protein standard from clofibrate-treated male rat liver; lane 2, 50 µg of protein from homogenates of isolated mesenteric arteries from rats on LS diet; and lane 3, 50 µg of protein from homogenates of isolated mesenteric arteries from rats on a HS diet. C: pixel densities of 51-kDa CYP450-4A immunoreactive protein band. D: pixel densities of 85-kDa glycosylated immunoreactive protein band. Values in C and D are expressed as percentage of the value of the microsomal standard on the same gel; *P < 0.05, significant change compared with LS controls; n = 6.

 
Effects of HS diet and CYP450-4A {omega}-hydroxylase inhibition on vasoconstrictor responses to NE. In control experiments (before DDMS), there was no significant difference in NE sensitivity in mesenteric arteries from rats fed a LS or HS diet (Fig. 3). This observation is consistent with previous reports that show similar NE sensitivity in skeletal muscle resistance arteries from Sprague-Dawley rats fed LS and HS diets (41). Inhibition of CYP450-4A {omega}-hydroxylase with DDMS had no effect on resting diameter or maximal response of the vessels to 10 µM NE in either group. However, DDMS produced a significant rightward shift of the NE concentration-response curve and reduced the magnitude of the response to 0.1 and 1 µM NE in vessels obtained from rats fed either a LS or a HS diet, respectively (Fig. 3, A and C). In the presence of DDMS, the EC50 for NE was significantly higher (P < 0.05) in arteries obtained from rats fed a HS diet (1.82 ± 0.22 µM) than in vessels obtained from rats fed a LS diet (1.22 ± 0.08 µM). In a separate series of time-control experiments, NE sensitivity was unaffected by repeating the NE concentration-response curve in vessels from rats on a LS or HS diet (Fig. 3, B and D, respectively).



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  		Fig. 3. Norepinephrine (NE)-induced constriction of mesenteric arteries from normotensive Sprague-Dawley rats on LS and HS diets. A and B: rats on LS diet for 3 days. C and D: rats on HS diet for 3 days. A and C: responses to NE before and after inhibition of CYP450-4A {omega}-hydroxylase with 30 µM N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). B and D: responses during successive NE concentration-response curves in time-control experiments conducted in the absence of DDMS. Data are expressed as percent maximum constriction (means + SE); n = 6 LS and 6 HS in DDMS experiments and n = 8 LS and 7 HS in time-control experiments. *P < 0.05, significant difference in EC50 after DDMS treatment; {dagger}P < 0.05, significant reduction in response to a given concentration of NE before and after DDMS treatment. There were no significant differences in the time-control experiments in either group.

 
Effects of HS diet and CYP450-4A {omega}-hydroxylase inhibition on vessel responses to reduced PO2. Figure 4 summarizes the effects of blocking 20-HETE formation with DDMS on the response to hypoxia in NE (1 µM)-precontracted mesenteric arteries obtained from rats fed a LS or HS diet. In the control period, vessels obtained from rats fed a LS diet dilated in response to reduced PO2, whereas vessels obtained from rats fed a HS diet did not relax in response to reduced PO2. Inhibiting the formation of 20-HETE had no effect on the dilation of arteries from rats fed a LS diet, but it restored the dilation to hypoxia in arteries obtained from rats fed a HS diet.



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  		Fig. 4. Responses of NE (1 µM)-precontracted mesenteric resistance arteries from Sprague-Dawley rats to simultaneous reduction of perfusion and superfusion solution O2 concentration from 21 to 0% O2 before and after inhibition of CYP450-4A {omega}-hydroxylase with DDMS (30 µM). Data are expressed as mean changes in vessel diameter from 21% O2 control value in vessels from rats on LS (n = 5) and HS (n = 7) diets. Inner diameters of the arteries under resting conditions were 230 ± 15 µm in the LS group and 235 ± 10 µm in the HS group. Diameters of NE-preconstricted vessels were 138 ± 15 µm in the LS group and 150 ± 11 µm in the HS group. *P < 0.05, significant difference between responses in LS and HS groups; {dagger}P < 0.05, significant increase in magnitude of hypoxic dilation after inhibition of CYP450-4A {omega}-hydroxylase with DDMS.

 

   DISCUSSION
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Formed from arachidonic acid by CYP450-4A {omega}-hydroxylase, 20-HETE has been shown to contribute to the contractile response of several vasoconstrictor agents, e.g., ANG II (1, 8) and phenylephrine (45), and to vasoconstriction in response to elevations in PO2 (16, 21, 22) and transmural pressure (11, 13). Because a variety of evidence indicates that a HS diet alters vascular responses to vasodilator (2, 12, 24, 25, 27, 36, 42, 43) and some vasoconstrictor (41) stimuli independent of changes in arterial blood pressure, the present study was conducted to determine whether changes in dietary salt intake affect the expression of the CYP450-4A enzymes responsible for the formation of 20-HETE in mesenteric arteries obtained from Sprague-Dawley rats fed a LS or HS diet for 3 days. We also evaluated the role of 20-HETE in determining NE-induced constriction and hypoxic dilation of vessels of rats fed a HS or LS diet.

One of the major findings of the present study is that an increase in dietary salt intake leads to changes in the isoforms of CYP450-4A expressed in mesenteric arteries (see Table 1 and Fig. 1). In mesenteric arteries of rats on a LS diet, only mRNA for CYP450-4A3 could be detected by PCR. In contrast, mRNA for the CYP450-4A2, -4A3, and -4A8 isoforms could readily be detected in mesenteric arteries isolated from rats fed a HS diet. Of particular interest is the finding that the pattern of CYP450-4A {omega}-hydroxylase enzyme expression changed very rapidly in rats, i.e., after only 3 days on a HS diet. In addition to changes in the expression of mRNA for specific CYP450-4A {omega}-hydroxylase isoforms, a HS diet increased the expression of the 51-kDa immunoreactive protein band for CYP450-4A {omega}-hydroxylase in mesenteric arteries (see Fig. 2B), although expression of the 85-kDa immunoreactive band corresponding to the glycosylated form of these proteins (21) was not significantly altered. The finding that most of the enzyme appears to be in the glycosylated form in these experiments is consistent with the results of earlier studies (21), although the effect of glycosylation on CYP450-4A {omega}-hydroxylase enzyme activity is unknown.

Our previous studies demonstrated that the CYP450-4A3 and -4A8 isoforms are consistently expressed in cremasteric arterioles of Sprague-Dawley rats fed a normal-salt diet (37). The lack of expression of the CYP450-4A8 isoform in mesenteric arteries of rats fed a LS diet and the emergence of the CYP450-4A2 and -4A8 isoforms in vessels of animals fed a HS diet demonstrate that the pattern of isoform expression is highly regulated and varies greatly after changes in dietary salt intake.

Previous studies in kidneys of Sprague-Dawley rats demonstrated that the CYP450-4A1, -4A2, and -4A3 isoforms are all present in preglomerular arterioles, and that the major expressed isoform is CYP450-4A2 rather than -4A1 (39). However, treatment of renal microvessels with antisense oligonucleotides for the CYP450-4A1 isoform still causes a substantial reduction in 20-HETE production by the vessels (39). This indicates that CYP450-4A1, which has a very low expression but the highest catalytic activity, is the major 20-HETE-forming enzyme in renal microvessels. This observation raises the possibility that very small changes in expression of other isoforms, particularly the CYP450-4A1 isoform, could occur in response to a HS diet and affect the responses of the vessels to vasoactive stimuli in these experiments. This possibility is worthy of further investigation especially under conditions of more prolonged exposure to a HS diet.

The effects of a HS diet on vasoconstrictor responses to NE remain controversial. For example, one study (34) indicated that a HS diet increases aortic sensitivity to NE (associated with an increased blood pressure), whereas another study reported that neither short- nor long-term exposure to a HS diet had any effect on the response of skeletal muscle resistance arteries to NE (41). In the present study, we found that short-term exposure to elevated dietary salt intake, which leads to a profound impairment of vascular relaxation mechanisms in arterioles and isolated cerebral and skeletal muscle resistance arteries (12, 25, 27, 36), did not alter the vasoconstrictor responses to NE in mesenteric resistance arteries.

In these experiments, inhibition of 20-HETE formation with DDMS significantly reduced vasoconstrictor responses to NE and shifted the NE concentration-response curve to the right in arteries obtained from rats fed either a LS or a HS diet (see Fig. 3, A and C). This finding is consistent with previous reports that the CYP450-4A {omega}-hydroxylase-20-HETE system exists in mesenteric arteries (3, 40) and plays a role in modulating their response to vasoconstrictor agonists (3, 40, 45). In the presence of DDMS, the EC50 value describing NE sensitivity was significantly higher in arteries from rats on a HS diet than in DDMS-treated vessels from animals on a LS diet, which indicates that NE sensitivity is lower in arteries from rats on a HS diet after inhibition of 20-HETE formation. The latter observation suggests that 20-HETE plays a greater role in modulating NE-induced constriction in vessels from rats on a HS diet.

The changes in CYP450-4A isoform expression in vessels from animals on a HS diet suggest that the DDMS-sensitive component of the vasoconstrictor responses to NE in mesenteric resistance arteries of the rats fed a HS diet may be attributed to upregulation of the expression of the CYP450-4A2 and -4A3 isoforms and possibly the CYP450-4A8 isoform, although the contribution of the CYP450-4A8 isoform to the regulation of vascular tone remains unclear (33) as does the contribution of changes in CYP450-4A1 isoform expression that are below the limit of detection. In contrast, it appears that only the CYP450-4A3 isoform contributes to the DDMS-sensitive component of NE-induced constriction in vessels from rats on a LS diet.

In contrast to the lack of certainty regarding the effect of a HS diet on NE-induced vasoconstriction, a substantial amount of evidence has accumulated to indicate that elevated dietary salt intake leads to impaired relaxation of arterioles (2, 9, 25), cerebral and skeletal muscle resistance arteries (12, 24, 27, 36), and conduit vessels such as the aorta (46) in response to vasodilator stimuli including reduced PO2. In the present study, we demonstrated for the first time that hypoxic dilation is also significantly impaired in mesenteric arteries from rats fed a short-term HS diet.

An intriguing observation in this study was that the impaired hypoxic dilation in arteries from rats on a HS diet was restored by inhibiting 20-HETE production with DDMS, whereas hypoxic dilation of vessels from rats on a LS diet was unaffected by DDMS. The observation that inhibiting 20-HETE formation with DDMS unmasks a dilation to reduced PO2 in mesenteric arteries of rats on a HS diet is consistent with the results of a recent study by Kerkhof et al. (20), who reported that inhibition of CYP450 enzymes with 17-octadecynoic acid unmasks a large hypoxic dilation in isolated arterioles from the rat cremaster muscle. The finding that CYP450-4A enzyme inhibition with DDMS selectively restores hypoxic dilation in arteries from animals on a HS diet but does not affect the hypoxic dilation in arteries from rats on a LS diet suggests that the impaired dilation to hypoxia in vessels from rats on a HS diet may be linked to an unusual increase in 20-HETE production. Such an increase in 20-HETE production could be caused by alterations of CYP450-4A isoform expression to favor the expression of enzyme isoforms with a greater catalytic ability to produce 20-HETE (see Fig. 1 and Table 1) or possibly an increased expression of enzyme protein (see Fig. 2, B and C). The hypothesis that increased levels of 20-HETE play a role in opposing hypoxic dilation of arteries from rats on a HS diet in the present experiments is consistent with the results of a recent study by Ren et al. (32), who demonstrated that inhibiting the action of 20-HETE with its antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid restored the bradykinin-induced dilation of rabbit efferent arterioles that is lost when the vessels are treated with indomethacin. Those authors proposed that the glomerulus or efferent arteriole releases 20-HETE to oppose the epoxyeicosatrienoic acid (EET)-dependent vasodilation that occurs in response to bradykinin when cyclooxygenase is blocked with indomethacin. The latter conclusion is consistent with the hypothesis that enhanced production of 20-HETE due to changes in the expression of the different isoforms (see Fig. 1 and Table 1) and/or an increase in enzyme protein expression (see Fig. 2C) overrides hypoxic dilation in arteries from rats on a HS diet.

The studies of Wang et al. (40) and Zhang et al. (45) suggested that the CYP450-4A1 isoform might contribute to the development of hypertension in spontaneously hypertensive rats by producing more 20-HETE and sensitizing mesenteric arteries to vasoconstrictor substances such as phenylephrine. The absence of the expression of detectable amounts of CYP450-4A1 mRNA in mesenteric arteries of Sprague-Dawley rats fed a HS diet in the present study may be related to the absence of hypertension and may be reflected in the relatively small shift of the NE concentration-response curve after DDMS treatment. The question of whether long-term salt loading could lead to further differences in the expression of CYP450-4A isoforms (including CYP450-4A1 expression) and to overall changes in vasoconstrictor sensitivity that are different from those occurring in vessels of rats on a short-term HS diet is worthy of further investigation. Nonetheless, the significant effects of a HS diet on the expression of CYP450-4A mRNA and protein and the differential effects of CYP450-4A {omega}-hydroxylase inhibition with DDMS on vessel responses to NE and hypoxia indicate that changes in the expression of these 20-HETE-producing enzymes resulting in increased production of 20-HETE may contribute to altered reactivity of resistance arteries to vasoactive stimuli in animals on a HS diet, independent of changes in blood pressure.

Although we believe that the enhanced effects of CYP450 enzyme inhibition on the responses to NE and hypoxia that we observed in vessels from animals on a HS diet most likely result from an increased production of 20-HETE due to the altered expression of CYP450-4A {omega}-hydroxylase isoforms and the increased expression of CYP450-4A enzyme protein in arteries from animals on a HS diet, there are a number of caveats to the conclusion that increased enzyme expression leads to increased formation of 20-HETE by vessels from animals on a HS diet. For example, changes in vessel responses to CYP450 {omega}-hydroxylase inhibition in animals on a HS diet could reflect not only changes in the production of 20-HETE itself but also changes in the expression and activity of other enzyme systems that could affect 20-HETE levels indirectly by metabolizing the compound, by indirectly affecting the expression or activity of CYP450-4A {omega}-hydroxylase, or by producing vasodilator compounds that oppose the vasoconstrictor effect of 20-HETE. In each of these cases, the effects of inhibiting CYP450 {omega}-hydroxylase would be amplified in vessels from animals on a HS diet. For example, 20-HETE can be converted into other vasoactive metabolites by cyclooxygenase and lipoxygenase (5, 30, 33), so that the enhanced effect of 20-HETE inhibition on vascular reactivity in arteries from animals on a HS diet could reflect reduced metabolism of 20-HETE to other compounds that either have vasodilator actions or lack vasoconstrictor activity. Epoxygenase compounds such as EETs can also oppose the vasoconstrictor actions of 20-HETE (17). Therefore, a reduced formation of EETs in the presence of unchanged levels of 20-HETE could lead to an enhanced effect of 20-HETE inhibition on NE-induced constriction or hypoxic dilation as seen in the present study. However, this seems to be unlikely in the view of the study of Oyekan et al. (31), which shows that 7 days of elevated dietary salt intake (2% NaCl) increased epoxygenase activity in the renal cortex and medulla (which would lead to increased levels of vasodilator EETs), but decreased {omega}-hydroxylase activity in the renal cortex.

Nitric oxide (NO) also modulates CYP450-4A {omega}-hydroxylase expression and 20-HETE synthesis (33) and appears to exert a tonic inhibitory influence on CYP450-dependent metabolism of arachidonic acid (30). Okeyan and McGiff (30) reported that inhibition of endogenous NO synthase unmasks an important vasoconstrictor effect of CYP450-dependent metabolites of arachidonic acid in kidneys. Under these conditions, higher levels of NO would reduce 20-HETE levels, whereas lower levels of NO would increase the influence of 20-HETE on vascular tone and vessel reactivity. Wilcox et al. (44) have shown that a HS diet augments NO production in kidneys, which could affect CYP450-4A enzyme expression and the levels of 20-HETE in tissue. However, this increase in NO production during a HS diet could reflect differences between the kidney and systemic vessels, because there is evidence that a HS diet leads to reduced NO production in aorta (46) and impaired NO-dependent dilation in middle cerebral arteries (36) and systemic microvessels (2). In the latter case, reduced NO release could in fact contribute to enhanced contribution of 20-HETE to vasoconstrictor responses in the vessels.

Finally, it is possible that vascular sensitivity to the constrictor effects of 20-HETE itself also may be increased by elevation of dietary salt content. Such an increase in 20-HETE sensitivity has been demonstrated in skeletal muscle arterioles (22) and mesenteric arteries (45) of spontaneously hypertensive rats vs. age-matched normotensive Wistar-Kyoto control animals, but the effects of a HS diet on vasoconstrictor sensitivity to 20-HETE remain to be determined.

Thus although changes in enzyme expression in animals on a HS diet [together with the previous demonstration that DDMS is a highly specific inhibitor of CYP450-4A {omega}-hydroxylase that results in reduced 20-HETE formation by the enzyme (38)] suggest that the 20-HETE-dependent changes in vessel reactivity in animals on a HS diet are due to increased formation of the compound by CYP450-4A {omega}-hydroxylase, the considerations discussed here emphasize the importance of directly measuring 20-HETE production in resistance arteries of animals on a HS diet. Other areas of investigation such as evaluating the effects of inhibiting cyclooxygenase 1 and 2 and/or NO synthase on the proposed actions of 20-HETE in these vessels could also provide important insights into the factors that determine the contribution of altered 20-HETE formation to changes in vascular reactivity during exposure to elevated dietary salt intake.

In summary, the present study shows that a HS diet leads to upregulated expression of CYP450-4A mRNA and increases the expression of CYP450-4A {omega}-hydroxylase protein in mesenteric resistance arteries of Sprague-Dawley rats. The predominant isoform expressed in rats fed a LS diet is CYP450-4A3, whereas the CYP450-4A2, -4A3, and -4A8 isoforms are all expressed in vessels obtained from rats fed a HS diet. Inhibition of 20-HETE formation with DDMS reduces the sensitivity of mesenteric arteries to the vasoconstrictor effects of NE, and the inhibition of NE-induced constriction by DDMS in arteries from rats on a HS diet is greater than that in vessels obtained from rats fed a LS diet. DDMS also restores the impaired dilation in response to reduced PO2 in rats fed a HS diet. Taken together, these data suggest that elevated dietary salt intake alters vascular CYP450-4A expression and 20-HETE-dependent mechanisms of vascular control in mesenteric resistance arteries. These findings raise the possibility that the CYP450-4A {omega}-hydroxylase system may contribute to altered vascular control mechanisms in salt-sensitive hypertension and could also affect vascular regulation in normotensive individuals on HS diets.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants HL-29587, HL-65289, HL-72920, and DK-38226 and by the Robert A. Welch Foundation.


   ACKNOWLEDGMENTS
 
The authors thank Tianjian Huang for valuable technical assistance in the present study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Lombard, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: jlombard{at}mcw.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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