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Vascular Physiology Group, Departments of 1Cell Biology and Physiology and 2Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Submitted 6 December 2002 ; accepted in final form 27 February 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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630 Torr) and CH (PB, 380
Torr for 48 h). VSM cell Em was normalized between groups
following administration of the CYP inhibitors 17-octadecynoic acid and
SKF-525A. VSM cell hyperpolarization after CH was not altered by
cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor
sulfaphenazole normalized VSM cell Em between groups.
Iberiotoxin also normalized VSM cell Em, which suggests
that large-conductance, Ca2+-activated K+
(BKCa) channel activity is increased after CH. Sulfaphenazole
administration restored phenylephrine-induced and myogenic vasoconstriction
and Ca2+ responses of mesenteric resistance arteries
isolated from CH rats to control levels. Western blot experiments demonstrated
that CYP2C9 protein levels were greater in mesenteric arteries from CH rats.
In addition, 11,12-EET levels were elevated in endothelial cells from CH rats
compared with controls. We conclude that enhanced CYP2C9 expression and
11,12-EET production following CH contributes to BKCa
channel-dependent VSM cell hyperpolarization and attenuated
vasoreactivity.
rat; epoxyeicosatrienoic acid; endothelial-derived hyperpolarizing factor; ratiometric calcium measurement; mesenteric circulation
Endothelial-derived metabolites of arachidonic acid are potent vasodilator and VSM cell hyperpolarizing factors (11, 33). For example, the cyclooxygenase product prostacyclin activates ATP-sensitive K+ (KATP) channels to alter VSM resting membrane potential (Em; Ref. 17), whereas the cytochrome P-450 (CYP) epoxygenase products epoxyeicosatrienoic acids (EETs) activate large-conductance Ca2+-activated K+ (BKCa) channels (3). We hypothesized that increased cyclooxygenase and/or CYP epoxygenase activity contribute to endothelium-dependent VSM cell hyperpolarization and attenuated vasoconstrictor reactivity following CH. Our findings suggest that enhanced expression of a CYP isoform 2C9 (CYP2C9)-like protein and increased EET production contribute to endothelium-dependent VSM cell hyperpolarization and blunted sensitivity to vasoconstrictor stimuli following prolonged hypoxic exposure.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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630 Torr).
Our laboratory previously reported that the arterial PO2
for rats under control conditions was 73 ± 1 compared to 44 ± 2
Torr under hypoxic conditions
(20). The duration of hypoxic
exposure employed for this study was selected based on previous reports that
demonstrate attenuated vascular reactivity following 48-h hypoxic exposure for
the renal and mesenteric circulations in vivo
(20,
23). In addition, attenuated
reactivity of mesenteric and diaphragmatic small arteries isolated from rats
exposed to hypoxia for 48 h has also been reported
(8,
9,
20,
38). Immediately before
experimentation, rats were deeply anesthetized with pentobarbital sodium (32.5
mg ip); after vessels were harvested, rats were killed by exsanguination.
VSM cell resting Em. VSM cell
Em values were recorded from superior mesenteric artery
(SMA) strips using glass intracellular microelectrodes. The chests and
abdomens of anesthetized rats were opened, and heparin (100 U in 0.1 ml) was
injected into the hearts to prevent clotting. SMAs were isolated and excised,
and artery strips were secured in an organ bath with the luminal surfaces
exposed. Strips were superfused (5 ml/min) with physiological saline solution
(PSS) that contained (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19
NaHCO3, 1.8 CaCl2, and 5.5 glucose warmed to 37°C
and aerated with a normoxic gas mixture that consisted of 21% O2-6%
CO2-73% N2. VSM cells were impaled with microelectrodes
(tip resistance, 100 M
) filled with 3 M KCl inserted into the
artery strip through the endothelial surface. A Neuroprobe model 1600
amplifier (A-M Systems) was used to record membrane potential. Analog output
from the amplifier was low-pass filtered at 1 kHz and routed to a Tektronix
RM502A oscilloscope and a Gould chart recorder. Criteria for acceptance of
Em recordings were 1) an abrupt negative
deflection of potential as the microelectrode was advanced into a cell,
2) stable Em for at least 3 min, and
3)an abrupt change in potential to 0 mV after the electrode was
retracted from the cell. Generally, recordings from several VSM cells were
made for each animal. The mean potential of all VSM cells recorded for a
particular rat was considered as a single replicate for statistical
purposes.
VSM cell Em values were recorded for vessels isolated from control and CH rats in the presence of vehicle, the nonselective CYP inhibitors 17-octadecynoic acid (17-ODYA, 1 µM) and SKF-525A (5 µM), the cyclooxygenase inhibitor meclofenamate (10 µM), or the selective CYP2C9 inhibitor sulfaphenazole (10 µM); n = 5 animals for each group. In additional experiments, Em measurements from VSM cells in SMAs isolated from control and CH rats were recorded in the presence of the BKCa channel inhibitor iberiotoxin (50 nM) or vehicle (n = 5 animals for both control and CH groups). SKF-525A and 17-ODYA were dissolved in ethanol, whereas sulfaphenazole, meclofenamate, and iberiotoxin were dissolved in PSS. All experiments were performed under normoxic conditions to eliminate the hyperpolarizing effects of acute hypoxia (19).
Isolated vessel preparation. Mesenteric resistance arteries from control and CH rats were isolated and pressurized. The mesenteric arcade was excised and transferred to ice-cold dissecting solution that contained 3 mM MOPS (pH 7.4), 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2.5 mM CaCl2, 1 mM KH2PO4, 0.02 mM EDTA, 2 mM pyruvate, 5 mM glucose and 1% bovine serum albumin. The arcade was secured in a Silastic-coated petri dish that contained dissecting solution. Resistance-artery branches were cleaned of adipose tissue and transferred to a beaker of dissecting solution. Fourth- or fifth-order vessel segments [passive inner diameter (ID) at 70 Torr, 100–200 µm] were dissected from the cleaned branches, transferred to a vessel chamber (Living Systems), cannulated with glass micropipettes, and secured with ligatures and blood was gently rinsed from the lumen. Vessels were slowly pressurized to 70 Torr with PSS using a servo-controlled peristaltic pump (Living Systems) and were superfused (5 ml/min) with warmed (37°C) PSS aerated with a normoxic gas mixture (21% O2-6% CO2-73% N2). After a 30-min equilibration period, intraluminal pressure was slowly increased to 120 Torr, vessels were stretched to remove bends, and pressure was reduced to 70 Torr for an additional 30-min equilibration period. Pressurized resistance arteries were loaded with the cell-permeant ratiometric Ca2+-sensitive fluorescent dye fura 2-AM (Molecular Probes). Fura 2-AM was dissolved in anhydrous DMSO at a concentration of 1 mM. Immediately before loading, fura 2-AM was mixed with 0.5 volumes of a 20% solution of Pluronic acid in DMSO, and this mixture was diluted with dissecting solution to yield a final concentration of 2 µM fura 2-AM and 0.05% Pluronic acid. Vessels were incubated in this solution for 45 min at room temperature in the dark. Administration of fura 2 to the abluminal surfaces of pressurized arterioles has been shown to preferentially load VSM cells (25). Before experimentation, vessels were equilibrated for 20 min with warmed, aerated PSS following the loading period to wash out excess dye and allow for esterification of AM groups.
Myogenic and agonist-induced vasoconstrictor and
Ca2+ responsiveness. Myogenic and phenylephrine
(PE)-induced vasoconstrictor responses were determined for small mesenteric
arteries isolated from control and CH rats. Fura-loaded vessels were exposed
to a series of pressure steps between 20 and 120 Torr, and spontaneous
myogenic tone was allowed to develop at each step for 3 min. After
pressure-response curves were completed, vessels were reequilibrated at 70
Torr for 10 min and were then superfused with PSS that contained increasing
concentrations of PE (0.05–50 µM). The ID was continuously monitored
using video microscopy and edge-detection software (IonOptix). Fura-loaded
vessels were alternatively excited at 340 and 380 nm at a frequency of 10 Hz,
and the respective 510-nm emissions were quantified using a photomultiplier
tube and recorded using IonWizard software (ver. 4.4; IonOptix). Vessel wall
[Ca2+]i was calculated at each pressure as
the mean F340/F380 ratio from the background-subtracted
510-nm signal collected over 3 min. After the pressure-response and PE
curves were completed, intraluminal pressure was maintained at 70 Torr, and
vessels were superfused for 1 h with Ca2+-free PSS that
contained (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19
NaHCO3, 5.5 glucose, and 3 EGTA. Another pressure-response curve
was completed for vessels under Ca2+-free conditions to
obtain passive responses. Myogenic tone was calculated as the percent
difference of ID observed for Ca2+-containing vs.
Ca2+-free PSS at each pressure. PE-induced
vasoconstriction was calculated as the percent change in ID relative to the ID
in Ca2+-free PSS at an intraluminal pressure of 70 Torr.
Vasoconstrictor and Ca2+ responses were determined for
arteries from control and CH rats in the presence of sulfaphenazole (10 µM)
or vehicle (n = 6 animals for each group). All experiments were
performed under normoxic conditions to eliminate the vasodilatory effects of
acute hypoxia (18).
Effects of exogenous 11,12-EET administration. Fura-loaded
mesenteric small arteries were preconstricted with PE to 30% of basal
diameter, and the effects of 11,12-EET (1 µM) administration on diameter
and vessel wall [Ca]i were determined.
Western blot analysis for CYP2C9. Arteries from the entire mesenteric arcade were isolated from control and CH rats and were snap-frozen in liquid nitrogen. Vessels were homogenized on ice in 10 mM Tris · HCl buffer (pH 7.4) that contained 255 mM sucrose, 2 mM EDTA, 12 µM leupeptin, 4 µM pepstatin A, 1 µM aprotinin, and 2 mM PMSF (all from Sigma). Homogenates were centrifuged at 13,400 g at 4°C for 10 min to remove tissue debris, and the microsomal fraction was collected by centrifugation at 100,000 g for 1 h. Protein concentrations of samples were determined using the Bradford method (Bio-Rad protein assay). Microsomal proteins were resolved on 7.5% SDS-PAGE minigels (Bio-Rad). In addition to the samples, each gel included both molecular mass (Bio-Rad) and a CYP2C-positive control that consisted of microsomal proteins from rat liver (16, 35). Separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked with 5% nonfat milk, 3% BSA (Sigma), and 0.05% Tween 20 (Bio-Rad) in Tris-buffered saline solution [TBS; 10 mM Tris · HCl and 50 mM NaC1 (pH 7.5)]. Blots were incubated overnight at room temperature with an anti-CYP2C9 antibody (R&D Systems) diluted 1/200 in TBS that contained 5% nonfat milk. According to the supplier, this antibody binds to the rat CY2C isoform that is homologous to the human epoxygenase CYP2C9. Immuno-chemical labeling was achieved by incubation for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; StressGen) in TBS, which was followed by enhanced chemiluminescence labeling (ECL; Amersham). Protein bands were detected by exposure to chemiluminescence-sensitive film (Kodak). Exposed films were digitized, and densitometric analysis of protein bands was performed using Scion Image software (version 4.0.2). Numerical densitometry units were compared between groups. Membranes were stained with Coomassie brilliant blue following immunoblotting procedures to confirm equal protein loading per lane.
EET levels in vascular tissue. SMAs were isolated from control and CH rats. Endothelial cells were harvested from these vessels after intraluminal incubation with dispase (2.4 U/ml) for 45 min at 37°C (31). Endothelial cells recovered from two arteries (from different rats) were pooled and then homogenized in 0.2 M KCl that contained 0.01% butylated hydroxytoluene (BHT; Ref. 13). PGB1 (100 ng) was added as an internal standard to each sample to control for extraction efficiency (24). Endothelial cell homogenates were extracted three times with an equal volume of diethyl ether that contained 0.005% BHT and 0.1% glacial acetic acid, and the recovered organic phase was evaporated under nitrogen (13). Dried samples were dissolved in equilibration buffer and subjected to reverse-phase HPLC with on-line photodiode array detection using a modification of a previously described method (24). Endothelial homogenates were separated on an analytical HPLC column (2 x 250 mm) packed with ODS-Hypersil (particle size, 5 µM; pore size, 120 Å). The mobile phase used consisted of acetonitrile, methanol, and water (59:7:34, vol/vol/vol) supplemented with 0.0001 volume of 88% formic acid with 6 mM pentanesulfonic acid and 20 µM EDTA. The pH of the mobile phase was adjusted to 4.36 with 10% aqueous solution of triethylamine. Flow rate was 0.25 ml/min. The column was kept at 40°C during separation.
CYP epoxygenase products in endothelial cell homogenates were identified by comparison with the retention time and spectral plot of authentic EET standards (Cayman Chemical). CYP epoxygenase products were measured in vessels isolated from control (n = 4) and CH rats (n = 4). In addition, EET levels were determined for CH rat endothelial cells harvested from vessels that had been incubated in the presence of sulfaphenazole (10 µM) for 1 h at 37°C (n = 5).
CYP2C9 enzyme activity. The effects of the CYP2C9 inhibitor
sulfaphenazole and zinc protoporhryin IX (ZnPPIX) on CY2C9 activity in vitro
were examined by monitoring the conversion of dibenzylfluorescein (DBF) to
flourescein by microsomes isolated from baculovirus-infected insect cells
(cell line BTI-TN-5B1-4) that overexpressed CYP2C9 as well as P-450
reductase and cytochrome b5 (Gentest). CYP2C9 microsomes
were incubated in a buffer that consisted of 50 mM
KH2PO4/K2HPO4 (pH 7.4), 1 mM
MgCl2, 1 mM NADPH, 10 mM isocitrate, and 0.2 U/ml isocitrate
dehydrogenase (all from Sigma) and 2 µM DBF (Gentest) for 30 min at
37°C. Reactions were performed in the presence of sulfaphenazole (10
µM), ZnPPIX (500 nM), or vehicle (n = 5 for all groups). The
reactions were terminated by the addition of NaOH to a final concentration of
0.5 M, and the samples were incubated at 37°C for an additional 2 h. The
relative amount of fluorescein generated by each reaction was quantified using
a fluorescence spectrophotometer [excitation wavelength (
), 485 nm;
emission , 538 nm].
Calculations and statistics. All data are expressed as means ± SE. Values of n refer to the number of animals in each group. Two-way ANOVA followed by Student-Newman-Keuls post hoc test where appropriate was used to make comparisons for most experiments. Unpaired t-tests were used to make comparisons of Western blot densitometric analyses between control and CH groups. One-way ANOVA followed by Student-Newman-Keuls post hoc test was used to compare 11,12-EET levels between groups and the effects of sulfaphenazole and ZnPPIX on CYP2C9 activity in vitro. A probability of ≤0.05 was accepted as statistically significant for all comparisons.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Effects of BKCa channel inhibition on VSM cell membrane potential. Previous studies have demonstrated that CYP-derived EETs elicit VSM cell hyperpolarization by activating BKCa channels (3). We therefore examined the effects of the specific BKCa channel inhibitor iberiotoxin on VSM cell Em in SMAs from control and CH rats. Iberiotoxin administration did not alter VSM cell Em values in arteries from control rats (Fig. 3). However, VSM cells in iberiotoxin-treated vessels isolated from CH rats were depolarized compared with VSM cells in vehicle-treated vessels from CH rats (Fig. 3). In addition, VSM cell Em values did not differ between arteries from control and CH rats following iberiotoxin administration (Fig. 3).
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Effects of CYP2C9 inhibition on myogenic responsiveness. Consistent with previously reported findings (9), myogenic vasoconstrictor responsiveness was blunted in small arteries obtained from CH rats compared with controls at intraluminal pressures >20 Torr (Fig. 4). In addition, myogenic responsiveness of vessels from control rats treated with sulfaphenazole was not different from that of vehicle-treated vessels (Fig. 4A). In contrast, myogenic vasoconstriction of sulfaphenazole-treated vessels obtained from CH rats was greater than vehicle-treated vessels from this group (Fig. 4B). Furthermore, pressure-induced constrictor responses of vessels from CH rats were not different from those of control arteries after CYP2C9 inhibition (Fig. 4).
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These experiments also demonstrated that vessel wall [Ca2+]i was diminished in vessels obtained from CH rats compared with controls at intraluminal pressures >20 Torr (Fig. 5). Vessel wall [Ca2+]i of arteries from control rats that were administered sulfaphenazole was not different from vehicle-treated vessels from control rats (Fig. 5A), whereas vessel wall [Ca2+]i of sulfaphenazole-treated arteries isolated from CH rats was greater than that of vehicle-treated vessels from CH rats (Fig. 5B). These data also showed that vessel wall [Ca2+]i did not differ between arteries isolated from CH and control rats in the presence of sulfaphenazole (Fig. 5).
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Effects of CYP2C9 inhibition on PE-induced vasoconstriction. PE-induced vasoconstriction was attenuated for vessels isolated from CH rats compared with controls (Fig. 6; Ref. 8). In addition, PE-induced vasoconstriction was not different between sulfaphenazole- and vehicle-treated arteries isolated from control rats (Fig. 6A). However, PE-induced vasoconstriction of arteries from CH rats treated with sulfaphenazole was greater than that of vehicle-treated vessels isolated from CH rats (Fig. 6B). Furthermore, PE-induced vasoconstriction of CYP2C9-inhibited vessels from CH rats was not different from that of vehicle- or sulfaphenazole-treated control arteries.
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Changes in vessel wall [Ca2+]i that resulted from PE administration were not different for arteries isolated from control rats treated with sulfaphenazole or vehicle (Fig. 7A). In contrast, PE-induced vessel wall [Ca2+]i responses were greater for sulfaphenazole-treated arteries from CH rats compared with vehicle-treated vessels from CH rats (Fig. 7B). Vessel wall [Ca2+]i did not differ between arteries isolated from control and CH rats in the presence of sulfaphenazole.
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Effects of exogenous 11,12-EET administration. In agreement with an earlier report (27), 11,12-EET administration (1 µM) elicited dilation of PE-constricted mesenteric arteries. PE-induced constriction was reversed by 47.0 ± 13.6% (n = 3). In addition, 11,12-EET reversed PE-induced increases in vessel wall [Ca]i by 30.9 ± 11.4% (n = 3). The vehicle for 11,12-EET (ethanol) was without effect.
CYP2C9 protein levels. A protein homologous to CYP2C9 was detected
in microsomes prepared from mesenteric arteries isolated from both control and
CH rats as a single major band of 56 kDa
(Fig. 8A). The
electrophoretic mobility of this band was identical to that of a band detected
in a gel lane loaded with microsomes from rat liver, a tissue that has been
shown to express CYP2C proteins
(16,
35). CYP2C9-band density was
greater in lanes loaded with microsomes from CH rats compared with controls
(Fig. 8B).
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EET levels in vascular tissue. A peak that is consistent with the retention time and spectra characteristics of the 11,12-EET standard was detected in chromatograms of endothelial cells harvested from vessels isolated from both control and CH rats (Fig. 9, B and C). The area of the 11,12-EET peak normalized to the PGB1 internal standard for tissue from CH rats was greater than that of controls (Fig. 9E). In addition, these experiments demonstrated that the area of the 11,12-EET peak for vessels treated with sulfaphenazole after isolation from CH rats (Fig. 9D) was less than that of vehicle-treated vessels from CH rats (Fig. 9E).
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Effects of sulfaphenazole and ZnPPIX on CYP2C9 activity. A previous report from our laboratory demonstrated that VSM cell Em values in SMAs from CH rats were normalized to control levels by administration of the heme oxygenase (HO) inhibitor ZnPPIX (8). The current study reports similar findings for the CYP2C9 inhibitor sulfaphenazole (see Fig. 2). Because CYPs are heme-containing enzymes and may be inhibited by ZnPPIX, an in vitro assay was employed to examine the potential inhibitory effects of ZnPPIX and sulfaphenazole administration on CYP2C9 activity. Consistent with previous reports (26, 29), administration of sulfaphenazole (10 µM) nearly abolished CYP2C9 activity (Fig. 10). In contrast, CYP2C9 activity was not altered by ZnPPIX (500 nM) administration (Fig. 10).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Earlier reports from our laboratory indicate that blunted vasoconstrictor responsiveness following CH is associated with endothelium-dependent VSM cell hyperpolarization (8, 9). These previous findings also suggest that VSM cell hyperpolarization associated with prolonged hypoxic exposure is NOS independent (8), whereas the current study demonstrates that altered VSM cell Em following CH is also independent of cyclooxygenase activity (see Fig. 2). Endothelium-dependent VSM cell hyperpolarization that is independent of NOS and cyclooxygenase is attributed to EDHF (6). Various studies have reported that EDHF-type responses result from release of K+ from endothelial cells (10), myoendothelial gap junctional communication (5, 34, 37), and CYP epoxygenase activity (21). The current findings demonstrate that VSM cell Em is normalized between control and CH groups by inhibition of CYP activity (see Fig. 1), which suggests that products of this pathway contribute to VSM cell hyperpolarization that is associated with prolonged hypoxic exposure. Furthermore, administration of the selective CYP2C9 inhibitor sulfaphenazole also eliminated differences in VSM cell Em values between groups (see Fig. 2), which indicates that this isoform plays an important role in CH-associated VSM cell hyperpolarization. This observation is consistent with reports that show that agonist-induced NO- and prostacyclin-independent VSM cell hyperpolarization in porcine coronary arteries is mediated by CYP2C9-dependent production of EETs (2, 12–15, 21). Given that EETs can elicit VSM cell hyperpolarization through activation of BKCa channels (3), we examined the effects of BKCa channel blockade on VSM Em following CH. Selective inhibition of BKCa channels with iberiotoxin normalized VSM cell Em between control and CH rats (see Fig. 3), which suggests that hyperpolarization after prolonged hypoxia results from increased activity of this channel. Taken together, these findings support the hypothesis that enhanced CYP2C9 activity following CH contributes to VSM cell hyperpolarization via increased BKCa channel conductance.
VSM cell Em largely regulates vascular tone by determining the activity of voltage-dependent Ca2+ channels (28). In addition, vasoconstriction can be influenced by the sensitivity of the VSM cell contractile apparatus to [Ca2+]i (30). Previous findings from our laboratory have demonstrated that VSM cell hyperpolarization and attenuated vasoconstrictor responsiveness following CH are associated with decreased vessel wall [Ca2+]i, which demonstrates that blunted constriction results from diminished VSM cell [Ca2+]i rather than from altered Ca2+ sensitivity (8, 9). In agreement with these findings, the current study also shows that attenuated myogenic vasoconstriction is associated with decreased vessel wall [Ca2+]i. In addition, CYP2C9 inhibition enhances both myogenic and PE-induced constrictor responsiveness of vessels from CH rats without altering vasoconstriction of vessels from control rats. Furthermore, enhanced reactivity of vessels from CH rats was associated with increased vessel wall [Ca2+]i following sulfaphenazole administration, whereas neither vasoconstriction nor vessel wall [Ca2+]i of control vessels were significantly altered by CYP2C9 inhibition. Therefore, we conclude that a CYP2C9-dependent decrease in VSM cell [Ca2+]i rather than altered Ca2+ sensitivity contributes to blunted reactivity following CH. These findings suggest that CYP2C9 activity does not greatly influence vascular tone or vessel wall [Ca2+]i of arteries isolated from control rats.
Our findings demonstrate that CYP2C9 inhibition influences VSM cell Em and contractility of vessels isolated from CH rats without altering these variables in arteries from control rats. These preferential effects of CYP2C9 inhibition observed in vessels from CH rats may be due to increased expression or activity of CYP2C9 that result in elevated production of EETs or alternatively may be due to enhanced sensitivity of the vasculature to these compounds. To address these possibilities, we performed Western blots on mesenteric arteries isolated from control and CH rats. These experiments showed that expression of a protein that is immunologically consistent with CYP2C9 is increased in these tissues following CH exposure (see Fig. 8). This finding is in agreement with experiments that demonstrate elevated levels of 11,12-EET in SMA endothelial cells from CH rats compared with controls (see Fig. 9). These data also show that endothelial 11,12-EET levels in vascular tissue treated with sulfaphenazole after harvest from CH rats are not different from those of vehicle-treated controls, which suggests that a sulfaphenazole-sensitive mechanism is responsible for increased 11,12-EET production following CH (see Fig. 9). Our observations are consistent with reports that demonstrate increased 11,12-EET production associated with elevated CYP2C9 expression in porcine coronary arteries (13, 14). In addition, administration of 11,12-EET to preconstricted mesenteric arteries induced vasodilation and decreased vessel wall [Ca2+]i. We conclude that increased vascular 11,12-EET production associated with elevated CYP2C9 protein expression contributes to VSM cell hyperpolarization and attenuated vasoreactivity following prolonged hypoxic exposure. The enzymatic digestion procedures used to obtain cells for EET isolation do not preclude the possibility that endothelial homogenates could be contaminated by other cell types such as VSM cells. However, given that previous studies demonstrate that disruption of the endothelium normalizes VSM cell membrane potential between control and CH rats (8, 9) and that CYP2C9 is expressed by endothelial cells (12, 14, 15), we conclude that the endothelium is the most likely source of elevated 11,12-EET levels in vascular tissue from CH rats.
A previous study from our laboratory demonstrated that administration of the HO inhibitor ZnPPIX normalizes VSM cell Em between vessels isolated from control and CH rats (8) in a manner similar to CYP2C9 inhibition in the present study (see Fig. 2). The earlier report supports a role for endothelial-derived CO as a contributor to VSM cell hyperpolarization following CH, whereas the current study supports a role for 11,12-EET in this response. To investigate these potentially discordant findings, we tested the possibility that CYP2C9, a heme-containing enzyme, may be inhibited by ZnPPIX. However, ZnPPIX did not decrease the activity of CYP2C9 activity in vitro (see Fig. 10), which suggests that EET production is not diminished by this drug. These experiments do not rule out the possibility that ZnPPIX interferes with the EET-induced signal transduction pathway responsible for VSM cell hyperpolarization. Although the effects of sulfaphenazole on HO activity have not been reported, several studies suggest that this drug is very selective for CYP2C9 (13, 26, 29, 32). Sulfaphenazole reportedly does not alter VSM cell hyperpolarization that results from exogenously administered EETs, which suggests that unlike other CYP inhibitors, this drug does not have nonspecific effects that attenuate VSM cell hyperpolarization (13). Thus it appears possible that inhibition of either the HO or the CYP2C9 pathway is sufficient to normalize VSM cell Em in vessels from CH rats to control levels. Although further experimentation is required to establish a link between the pathways, these findings are consistent with the hypothesis that HO and CYP2C9 products cooperate to induce VSM cell hyperpolarization following CH. Potential mechanisms whereby these pathways may interact include stimulation of the activity of CYP2C9 or lipoxygenases that generate arachidonic acid by CO. Alternatively, the HO and CYP2C9 pathways may interact by influencing cellular redox status. For example, the HO product biliverdin and subsequently bilirubin are potent antioxidants (39), and inhibition of this pathway may result in a more reduced intracellular environment. In contrast, a recent study demonstrates that CYP2C9 activity generates functionally significant quantities of superoxide anions in porcine coronary endothelial cells (15). Furthermore, CYP activity may be inhibited by free radicals (36), which suggests that decreased levels of antioxidants may result in decreased EET production. Investigations of potential interactions between these two pathways are needed to elucidate the mechanism that is responsible for VSM cell hyperpolarization after CH.
In summary, we have demonstrated that CYP2C9 activity is associated with BKCa-dependent VSM cell hyperpolarization and attenuated vasoconstriction and Ca2+ responses following CH. In addition, our findings show that vascular CYP2C9 protein and EET levels are increased after prolonged hypoxic exposure. We conclude that elevated EET production that results from increased CYP2C9 expression contributes to endothelium-dependent VSM cell hyperpolarization and attenuated vasoreactivity following CH.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-58124 and HL-63207.
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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P ≤ 0.05 vs. vehicle-treated vessels
from CH rats; n = 5 rats/group.
