Renal 20-HETE inhibition attenuates changes in renal hemodynamics induced by L-NAME treatment in pregnant rats
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《美国生理学杂志》
Department of Physiology and Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta, Georgia
Department of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas
Department of Physiology, Medical College of Georgia, Augusta, Georgia, and Renal Department of Memorial Hospital, Sun Yat-Sen University, Guangdong Province, People’s Republic of China
ABSTRACT
We previously reported that inhibition of nitric oxide (NO) synthesis by N-nitro-L-arginine methyl ester (L-NAME) during late pregnancy leads to increased production of renal vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 (CYP) 4A-derived vasoconstrictor, in pregnant rats. However, the effect of upregulation of vascular 20-HETE production on renal function after NO inhibition is not known. To test the hypothesis that increased gestational vascular 20-HETE synthesis after NO inhibition is involved in mediating blood pressure and renal functional changes, we first determined the IC50 value of the effect of nitroprusside (SNP), a NO donor, on renal 20-HETE production in cortical microsomes. We then divided pregnant rats and age-matched virgin rats into a vehicle control group, an L-NAME treatment group (0.25 mg/ml in drinking water), and a group treated with L-NAME plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; CYP4A-selective inhibitor, 10 mg·kg–1·day–1 iv). After 4 days of treatment, we measured blood pressure, renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in each group. The addition of SNP (IC50 = 22 μM) decreased renal cortical 20-HETE production. In pregnant rats, L-NAME treatment led to significantly higher mean arterial pressure (MAP) and RVR, and lower RBF and GFR. Combined treatment with DDMS and L-NAME significantly attenuated the increases in MAP and RVR and the decrease in GFR, but not the reduction in RBF induced by L-NAME treatment. L-NAME and L-NAME plus DDMS had no significant impact on renal hemodynamics in virgin rats. In addition, chronic treatment with DDMS selectively inhibited cortical 20-HETE production without a significant effect on CYP4A expression in L-NAME-treated pregnant rats. In conclusion, NO effectively inhibits renal cortical microsomal 20-HETE production in female rats. In pregnant rats, the augmentation of renal 20-HETE production after NO inhibition is associated with increased MAP and RVR, whereas decreased GFR is negated by treatment of a selective and competitive CYP4A inhibitor. These results demonstrate that the interaction between renal 20-HETE and NO is important in the regulation of renal function and blood pressure in pregnant rats.
pregnancy; cytochrome P-450; arachidonic acid; eicosanoid; kidney; nitric oxide; hypertension
THE MAJOR CYTOCHROME P-450 (CYP)-derived eicosanoid in rat kidney, 20-hydroxyeicosatetraenoic acid (20-HETE), is formed primarily by CYP4A isoforms. The expression of CYP4A isoforms and production of 20-HETE in the small arterial vessels is well documented (12, 30, 34). In the microvessels, 20-HETE depolarizes vascular smooth muscle by inhibiting Ca2+-activated K+ channels and enhances the conductance of L-type Ca2+ channels (11, 40), thereby sensitizing vascular smooth muscle to constrictor stimuli and causing vasoconstriction (39). In the rat kidney, 20-HETE inhibits ion transport in the different segments of nephron. For example, in the renal tubules, 20-HETE inhibits Na+-K+-ATPase activity in a concentration-dependent manner (0.1 nM-1 μM) through phosphorylation of the -subunit of Na+-K+-ATPase by protein kinase C (25, 28). Escalante et al. (10) demonstrated the inhibitory effect of 20-HETE on sodium transport in the thick ascending limb of the loop of Henle (TALH). Interestingly, Wang and Lu (37), using a patch-clamp technique, demonstrated that 20-HETE blocks the 70-pS K+ channel in the apical membrane of TALH cells, thus limiting the amount of K+ available for transport via Na+-K+-2Cl– cotransporter and reducing the positive potential in the lumen, which is the main driving force for passive reabsorption of cations in the TALH. Because of these biological activities, 20-HETE has been linked to regulation of renal function and blood pressure in many animal models of hypertension (15, 20, 31).
Preeclampsia, which affects 5–10% of pregnancies in the U.S., is characterized by increased arterial blood pressure, generalized vasoconstriction, increased systemic resistance, widespread vascular endothelial damage, decreased fetal growth, and proteinuria (18). The exact mechanisms that mediate preeclampsia are still unknown. Several reports have suggested that nitric oxide (NO) may play an important role in its development (5, 19). Moreover, different investigators have demonstrated that chronic inhibition of NO synthesis in pregnant rats results in signs similar to those of preeclampsia (23, 38). Several studies have demonstrated that NO inhibits 20-HETE synthesis and interferes with 20-HETE vasoconstrictor activity in vivo (13, 27, 32). We previously demonstrated that NO binds differently to CYP4A1 and CYP4A3, the major renal CYP isoforms in female rats, and inhibits their catalytic activity (36). Moreover, the inhibition of NO production by N-nitro-L-arginine methyl ester (L-NAME) during late pregnancy in rats causes the augmentation of 20-HETE synthesis in renal microvessels (36). However, it is not known whether the upregulation of renal microvessel 20-HETE production affects renal function in L-NAME-treated pregnant rats.
In the present study, we examined the effect of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective CYP4A inhibitor, on various renal functional parameters, including mean arterial pressure (MAP), renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in pregnant rats treated with L-NAME during days 14-17 of gestation. Age-matched virgin rats served as controls. This study provides valuable information regarding the interaction between NO and 20-HETE in the regulation of renal function and blood pressure in pregnant rats.
MATERIALS AND METHODS
Materials. We obtained [1-14C]arachidonic acid (56 mCi/mmol) from DuPont-New England Nuclear (Boston, MA). All HPLC solvents and chemicals for buffer were obtained from Sigma (Milwaukee, WI). We purchased 20-HETE, epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) standard from Cayman Chemicals (Ann Arbor, MI).
Animals. All animals were purchased from Charles River Laboratories (Wilmington, MA). We conducted experiments in pregnant (timed pregnancy) and age-matched female Sprague-Dawley rats. All rats were maintained on a 12:12-h light-dark cycle and were housed two rats to a cage. All animal protocols were approved by the Institutional Animal Care and Use Committee and were in accordance with the protocols for animal use outlined in the Guide for the Care and Use of Laboratory Animals.
Protocol to evaluate the effect of sodium nitroprusside on renal microsomal 20-HETE production. We examined the stability of the NO donor sodium nitroprusside (SNP) with NO-sensitive litmus paper using Griess reagent [0.5 g of sulfanilamide plus 20 mg of N-(1-naphthyl)ethylenediamine dihydrochloride] dissolved in 10 ml of methanol (24). We preincubated SNP (15–56 μM, final concentration) with renal cortical microsomes (150 μg) isolated from female rats at 37°C for 20 min, then added [1-14C]arachidonic acid (0.4 μCi, 7 nmol), NADPH (1 mM), buffer (10 mM MgCl2 and 100 mM KH2PO4, pH 7.2) to the incubation mixtures in a final volume of 0.15 ml. Incubation was then carried out at 37°C for an additional 30 min. The reaction was terminated by acidification to pH 3.5–4.0 with 2 M formic acid. We extracted metabolites with ethyl acetate and determined 20-HETE production in the renal microsomes by HPLC as previously described (35). We also determined the IC50 value as previously described (33).
Protocol to evaluate the effect of DDMS on renal function in L-NAME-treated pregnant and virgin rats. We divided the pregnant rats (day 14 of gestation) and age-matched virgin rats into three groups (6 rats per group): 1) a vehicle-treated group (45% 2-hydroxypropyl--cyclodextrin), 2) an L-NAME treatment group; (0.25 mg/ml in drinking water), and 3) a group treated with L-NAME (0.25 mg/ml in drinking water) plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 10 mg·kg–1·day–1 iv). Each group was treated for 4 days. The dosages of L-NAME and DDMS were based on our previous studies and a literature search (4, 36). After treatment, we used these pregnant rats and age-matched female rats for a renal functional study. On day 18 of pregnancy, after finishing the functional study, we immediately removed their kidneys for renal cortical homogenate preparation to be used to assess renal 20-HETE production and CYP4A expression.
Renal functional measurements. We performed the renal functional study in pregnant rats and age-matched control female rats from the three treatment groups. Each rat was weighed before surgery. We anesthetized the rats with 2% isoflurane delivered by an anesthesia apparatus. Then, we placed one polyethylene cannula in the trachea (PE-205) to allow free breathing, one in the bladder (PE-240) to collect urine, one in the femoral artery (PE-50) for measuring and recording MAP with a pressure transducer, and one (PE-50) in the femoral vein for the infusion of agents. We then began infusing saline (3 ml/h iv) and a dose of 0.5 ml of FITC inulin (8 mg/ml in PBS, Sigma) was administered over 2 min as a priming dose. We performed a left laparotomy and placed a Transonic flow probe (Transonic System, Ithaca, NY) over the left renal artery to measure RBF. During the experiments, the rats’ body temperature was maintained at 37°C by a temperature controller (Cole Palmer Instrument) connected to a heating mat and a rectal temperature probe. After a volume of saline containing 6.2% BSA equal to 1.25% body wt had been infused, the intravenous infusion was switched to saline without BSA, but still with FITC inulin at 4 mg/ml. At least 45 min were allowed for equilibration following surgery before the beginning of the 30-min urine collections. Arterial blood (0.4 ml) was drawn from the femoral artery in the middle of each 30-min clearance period for measurement of GFR. An equal volume of normal saline was infused for volume replacement. MAP, RBF, and RVR were obtained from a computerized data collection and analysis system (EMKA Technologies, Falls Church, VA). We determined the concentration of FITC-inulin in plasma and urine using a GENios Plus fluorescent plate reader (Tecan, Research Triangle Park, NC) at 485-nm excitation and 538-nm emission. We used the concentration of FITC inulin in the plasma and urine to calculate GFR as described previously (29).
Isolation of renal cortical homogenates. After the renal functional study, we immediately isolated the kidneys from treated and control rats. Renal cortex from each rat was homogenized in 1 ml of 10 mmol/l potassium buffer (pH 7.7) containing 250 mmol/l sucrose, 1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonyl fluoride (PMSF), and 7.5 μl/ml protease inhibitor cocktail (Sigma). We centrifuged the homogenates for 15 min at 3,000 g and for 30 min at 11,000 g and collected supernatants and stored them at –80°C. The protocol for homogenate preparation was adapted from that of Hoagland et al. (14).
Arachidonic acid metabolism in renal cortical homogenates. We incubated renal cortical homogenates (1 mg) isolated from treated and control with [1-14C]arachidonic acid (0.2 μCi, 3.5 nmol) and NADPH (1 mmol/l, final concentration) in 1 ml potassium phosphate buffer (100 mmol/l, pH 7.4) containing 10 mmol/l MgCl2 for 15 min at 37°C. The reaction was terminated by acidification to pH 3.5–4.0 with 2 mol/l formic acid, after which arachidonic acid metabolites were extracted with ethyl acetate. We evaporated the ethyl acetate under nitrogen, resuspended the metabolites in 50 μl methanol, and injected them into the HPLC column. We performed reverse-phase HPLC on a 5-μm ODS-Hypersil column, 4.6 x 200 mm (Hewlett Packard, Palo Alto, CA) using a linear gradient of acetonitrile:water:acetic acid ranging from 50:50:0.1 to 100:0:0.1 at a flow rate of 1 ml/min for 30 min. The elution profile of the radioactive products was monitored by a flow detector (In/us System, Tampa, FL). We confirmed the identity of 20-HETE and DHETs with authentic standards. The activity of 20-HETE formation was estimated based on the specific activity of the added [1-14C]arachidonic acid and was expressed as pmoles per milligram of protein per minute as described previously (14, 35).
Western blot analysis. We separated renal cortical homogenates (10 μg) from treated and control rats by electrophoresis on a 10 x 20-cm, 8% SDS-polyacrylamide gel at 25 mA/gel at 4°C for 18–20 h. The proteins were transferred electrophoretically to an enhanced chemiluminescence (ECL) membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 10 mmol/l Tris·HCl, 0.1% Tween 20, and 150 mmol/l NaCl for 90 min, then washed three times with TBS. We incubated the membranes for 10 h with goat anti-rat CYP4A1 (1:2,000; Gentest, Woburn, MA) at room temperature, washed them several times with TBS, and incubated them again for 1 h with a 1:5,000 dilution of horseradish peroxidase-coupled rabbit anti-goat secondary antibody for CYP4A1. We developed the immunoblots using an ECL detection kit (Amersham, Arlington Heights, IL). To normalize the expression of CYP isoforms, we incubated renal cortical homogenates (10 μg) from treated and control rats with a 1:5,000 dilution of mouse anti-chicken -actin antibodies (Sigma, St. Louis, MO) for 10 h. The secondary antibody was horseradish peroxidase-coupled rabbit anti-mouse antibody (1:5,000). Immunoreactive -actin was detected as described earlier. We scanned the ECL films of Western blot analyses and performed densitometric analysis with Scion Image software using a gray color scale as a standard (SPSS 10.0).
Statistical analysis. Data are expressed as means ± SE. All data were analyzed by SPSS 10.0 computer software (SPSS, Chicago, IL). We used one-way ANOVA or Student’s unpaired two-tailed test for statistical analysis. Statistical significance was set at P < 0.05.
RESULTS
Effect of SNP on renal cortical microsomal 20-HETE production. To examine the stability of NO donor SNP, we incubated SNP (0.01 to 1 mM) at 37°C for varying times, then tested a drop of solution from each concentration with NO litmus paper prepared using filter paper that had been incubated with Griese reagent (24). As shown in Fig. 1A, SNP released NO (an orange spot shown by arrow bar) within 10–20 min of incubation. The litmus paper proved to be a convenient and useful method for qualitatively checking NO production and indicated that SNP stably released NO within 20 min at 37°C. To examine whether NO has an inhibitory effect on 20-HETE production in renal cortical microsomes, we incubated renal cortical microsomes isolated from female rats with SNP (15–56 μM) at 37°C for 20 min, then incubated them with arachidonic acid and NADHP and determined 20-HETE production by HPLC. As shown in Fig. 1B, the addition of SNP inhibited 20-HETE production in renal cortical microsomes in a concentration-dependent manner, with an IC50 of 22 μM.
Effects of DDMS on renal function of L-NAME-treated pregnant and control rats. The preceding study showed upregulation of renal vascular 20-HETE production after chronic treatment of pregnant rats with L-NAME (36). To assess the contribution of elevated 20-HETE production to renal hemodynamics after withdrawal of NO, we examined renal functional parameters in pregnant and age-matched virgin female rats treated with vehicle, L-NAME, or L-NAME plus DDMS. As shown in Fig. 2, MAP was significantly decreased on day 18 of gestation compared with that in control nonpregnant rats. L-NAME treatment from days 14 to 17 of gestation caused a significant elevation of blood pressure, from 97 ± 3 to 134 ± 4 mmHg. This elevation of MAP was attenuated by combined treatment with L-NAME and DDMS. RBF values were slightly increased in pregnant rats compared with virgin rats (6.1 ± 0.4 vs. 5.1 ± 0.4 ml/min). L-NAME treatment of pregnant rats significantly decreased RBF from 6.1 ± 0.4 to 3 ± 0.3 ml/min, whereas L-NAME plus DDMS treatment did not significantly alter RBF in L-NAME-treated pregnant rats. RVR values in pregnant rats were significantly lower than those in nonpregnant control rats (15.3 ± 1.7 vs. 21.3 ± 1.6 mmHg·ml–1·min–1, P < 0.05). L-NAME treatment of pregnant rats also caused a significant increase in RVR, from 15.3 ± 1.7 to 45.3 ± 4.0 mmHg·ml–1·min–1. Moreover, the elevation of RVR in response to L-NAME was attenuated by treatment with L-NAME plus DDMS. As shown in Fig. 3, the GFR values in pregnant rats were significantly higher than those in nonpregnant control rats (1.6 ± 0.1 vs. 1.1 ± 0.04 ml/min, P < 0.05). L-NAME treatment markedly decreased GFR in pregnant rats to the levels in nonpregnant rats. This reduction of GFR by L-NAME treatment was attenuated by treatment with L-NAME plus DDMS (Fig. 3). In contrast, the MAP, RBF, RVR, and GFR in L-NAME-treated virgin rats did not significantly differ from control rats (Figs. 2 and 3). Similarly, combined treatment with L-NAME and DDMS had no impact on the changes in MAP, RBF, RVR, and GFR in virgin rats (Figs. 2 and 3).
Effects of DDMS on renal cortical 20-HETE production of L-NAME-treated rats. To study the selectivity of the effect of DDMS on renal 20-HETE synthesis, we used HPLC to examine renal cortical arachidonic acid metabolism in pregnant rats treated with L-NAME and L-NAME plus DDMS. As shown in Fig. 4A, incubation of renal cortical homogenates isolated from L-NAME treatment with [14C]arachidonic acid and NADPH produced DHETs and 20-HETE. After treatment with DDMS plus L-NAME, the conversion of arachidonic acid to 20-HETE (-hydroxylase activity) decreased by 44%. DHETs production was not affected compared with L-NAME treatment (Fig. 4A). The -hydroxylase activity in the L-NAME treatment group was 49 ± 4 pmol·min–1·mg protein–1. Treatment with DDMS plus L-NAME caused a 44% reduction in this activity to 26 ± 3 pmol·min–1·mg protein–1 (n = 4, P < 0.05). The selective inhibition of 20-HETE by DDMS treatment was also observed in L-NAME plus DDMS-treated virgin rats (data not shown). To examine the effect of DDMS treatment on CYP4A expression, we conducted Western blot analysis for CYP4A in renal cortical homogenates isolated from pregnant rats treated with L-NAME and DDMS plus L-NAME. A representative Western blot of CYP4A isoforms after two treatments is shown in Fig. 4B. Renal cortical homogenate CYP4A protein levels were similar in rats treated with L-NAME and DDMS plus L-NAME.
DISCUSSION
Normal pregnancy in rats is associated with increases in GFR and RBF and reductions in RVR and MAP (1, 17). Many different investigators have suggested that NO plays an important role in the regulation of renal function and blood pressure in pregnant rats (18, 19). NO causes the relaxation of smooth muscle in blood vessels, and the mechanisms for the action of NO on blood pressure regulation during pregnancy may be attributable to the upregulation of NO synthesis in blood vessels. It is also possible that pregnancy-induced changes in hormonal background contribute to the upregulation of NO production. Supporting this hypothesis, a prior study by Chen et al. (7) showed that estrogen stimulates NO synthesis through endothelial NO synthase (eNOS). Another study by Binko and Majewski (6) showed that estrogen increases the expression of inducible NO synthase (iNOS) in the vascular smooth muscle of blood vessels. In addition, Conrad et al. (8) demonstrated that the binding of NO to hemoglobin is detected only in the blood of pregnant, not nonpregnant, rats and Alexander et al. (1) demonstrated that increased NO production in pregnant rats is associated with upregulation of neuronal NO synthase (nNOS) and iNOS in the kidneys. These findings support the notion that upregulation of the NO system may be important in regulating vascular tone and blood pressure during pregnancy. To investigate the role of NO on the regulation of functional changes during pregnancy, Kassab et al. (16) used L-NAME, a competitive inhibitor of NO synthase, to study the consequence of NO inhibition on blood pressure and systemic and renal hemodynamic changes in pregnant rats. In this study, L-NAME-induced hypertension in pregnant rats is associated with increased RVR and decreased RBF (16). However, the mechanisms whereby L-NAME causes these changes in renal hemodynamics are still not very clear.
One possibility is that chronic blockade of NO by L-NAME in pregnant rats results in the amplification of renal vasoconstrictor factors that cause the changes in renal function. In a previous study, we demonstrated a marked increase in renal vascular 20-HETE production after L-NAME treatment of pregnant rats (36). Because 20-HETE is a vasoconstrictor of renal arterioles, we hypothesize that this increased renal vascular 20-HETE synthesis after chronic NO inhibition may contribute to the alteration of renal function in L-NAME-treated pregnant rats. The essential findings of the present study, that selective inhibition of renal 20-HETE production in pregnant rats attenuates the changes in renal hemodynamics caused by L-NAME treatment, are in accord with that hypothesis. This conclusion is based on the observation that L-NAME-induced renal functional changes such as increased MAP, increased RVR, and decreased GFR are attenuated by treatment with DDMS, a selective inhibitor of 20-HETE production. Although we observed attenuation of the changes of renal hemodynamics by DDMS treatment in L-NAME-pregnant rats, this treatment cannot totally reverse the changes in renal hemodynamics, returning them back to the levels in control pregnant rats (Figs. 2 and 3), perhaps because of increases in the sensitivity of vasoconstriction systems other than 20-HETE after NO blockade. Molnar and Hertelendy (22) demonstrated that in rats at late pregnancy, acute treatment with L-NAME enhances the sensitivity of the pressor responses to angiotensin II, norepinephrine, and arginine vasopressin (22). Edwards et al. (9) showed that L-NAME treatment during mid to late pregnancy causes elevation in the level of plasma endothelian-1. However, further investigation is needed to determine whether these vasoconstriction systems are involved in the regulation of renal hemodynamics after chronic NO blockade.
Besides its action on vascular relaxation, NO inhibits CYP enzyme systems (21, 31). The mechanisms for the inhibition of CYP enzymes by NO are thought to be reversible binding between NO and the heme moiety of CYP isoforms and irreversible modification of CYP isoforms by NO (21). In this regard, Sun et al. (32) showed that the addition of NO donors to recombinant CYP4A2 leads to the binding of NO to the heme moiety of CYP4A2 (an increases in the visible light absorbance at 440 nm). Minamiyama et al. (21) showed that NO can irreversibly modify the cysteine residues of CYP proteins. More recently, we demonstrated that NO binds to the heme moiety of CYP4A1 and CYP4A3 isoforms, the major enzymes for renal 20-HETE production in female rats, and that the NO donor SNP inhibits both CYP4A1 and CYP4A3-catalyzed 20-HETE production (36). These results are consistent with the results shown in Fig. 1, that SNP constantly releases NO and that SNP inhibits renal 20-HETE production in female rats. These results and those from our previous study (36) provide solid biochemical evidence of the interaction between NO and CYP4A enzymes and this interaction results in the inhibition of 20-HETE production in the kidneys.
Several investigators demonstrated the functional implications of interaction between NO and 20-HETE in vivo. For example, Magdalena et al. (2) found that acute administration of DDMS attenuated the NO-mediated vasodilatory response in rat renal arterioles and that a reduction in MAP and RVR was mediated by NO donor. Another report by the same group demonstrated that inhibition of 20-HETE production by DDMS treatment contributes to the cerebral vasodilation response to NO in rats (3). In addition, Oyekan and McGiff (26) showed that acute inhibition of 20-HETE by 12,12-dibromododec-enoic acid, another selective inhibitor of 20-HETE production (33), attenuated acute L-NAME-induced reductions in RBF and GFR, as well as increases in MAP and RVR in male rats. More recently, Hercule et al. (13) demonstrated that the treatment of antisense oligodeoxynucleotides for CYP4A isoforms attenuates both the reduction in RBF and the increase in RVR induced by L-NAME treatment. Because we did not observe the significant renal hemodynamic changes in L-NAME and L-NAME plus DDMS-treated virgin rats (Figs. 2 and 3), the interaction between NO and CYP4A isoforms may be a unique and important mechanism in the regulation of renal function during pregnancy. In pregnant rats, the overproduction of NO in the kidneys (1) can lead to binding of NO to CYP4A isoforms in renal arterioles and mask the vasoconstrictive effect of 20-HETE during normal pregnancy. When chronic L-NAME treatment blocks NO production, NO no longer keeps the 20-HETE system under control resulting in the elevation of renal vascular 20-HETE production (36) and the amplification of renal vasoconstriction mechanisms that cause increased RVR and MAP (Fig. 2). More importantly, we showed that the increases in RVR and MAP induced in L-NAME-treated pregnant rats were attenuated by the inhibition of renal 20-HETE synthesis (Fig. 2). Taken together, the present study solidly demonstrates that the interaction between NO and 20-HETE has many functional implications with respect to the regulation of blood pressure and renal function in pregnant rats.
It is well recognized that 20-HETE is formed endogenously in various tissues and exerts potent biological effects on cellular functions. Studies of its role on cellular levels are impeded by the difficulty in selectively targeting its synthesis and effects. This difficulty arises from that fact that the commonly used CYP inhibitors such as 1-aminobenzotriazole and 1-octadecynoic acid do not selectively inhibit 20-HETE production (31). We previously characterized several selective inhibitors of 20-HETE production in renal microsomes (33). Among them, DDMS was found to be highly specific for 20-HETE production, with an IC50 value about 2 μM, whereas its IC50 value for EETs formation is 60 μM. In the present study, we used DDMS to block the arachidonic acid -hydroxylase pathway and found that DDMS specifically blocked 20-HETE production without significantly affecting DHETs formation in pregnant rats (Fig. 4A). In addition, chronic DDMS treatment did not affect the renal expression of CYP4A isoforms in L-NAME-treated pregnant rats (Fig. 4B), which suggests that DDMS is a competitive inhibitor. Similar results for the competitive inhibition of 20-HETE by chronic DDMS treatment have been reported in the literature (4). Thus these data demonstrate that the selective inhibition of 20-HETE production by DDMS in vivo will be a very useful tool for elucidating the effect of 20-HETE in mediating physiological functions.
In summary, we presented evidence that chronic inhibition of NO production by L-NAME amplifies a vasoconstrictor system operating through 20-HETE that contributes to the changes in renal hemodynamics following suppression of NO production in pregnant rats. This conclusion is supported by the chronic effect of DDMS, a selective inhibitor of 20-HETE production, which inhibits the renal response to L-NAME-treated pregnant rats. This study demonstrates that activation of the renal 20-HETE pathway after removal of NO in the kidneys during pregnancy affects the regulation of renal function and blood pressure and that interaction between NO and 20-HETE may have important functional implications during pregnancy.
GRANTS
This study was supported by National Institutes of Health (NIH) Grant R01-HL-70887 to M.-H. Wang and by NIH Grant DK-38226 and a grant from Robert A. Welch Foundation to J. R. Falck.
ACKNOWLEDGMENTS
The authors thank Dr. D. M. Pollock for providing the protocol for measuring GFR. The authors also thank J. Cole for editorial assistance.
FOOTNOTES
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.
H. Huang and Y. Zhou contributed equally to the development of this research study.
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Department of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas
Department of Physiology, Medical College of Georgia, Augusta, Georgia, and Renal Department of Memorial Hospital, Sun Yat-Sen University, Guangdong Province, People’s Republic of China
ABSTRACT
We previously reported that inhibition of nitric oxide (NO) synthesis by N-nitro-L-arginine methyl ester (L-NAME) during late pregnancy leads to increased production of renal vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 (CYP) 4A-derived vasoconstrictor, in pregnant rats. However, the effect of upregulation of vascular 20-HETE production on renal function after NO inhibition is not known. To test the hypothesis that increased gestational vascular 20-HETE synthesis after NO inhibition is involved in mediating blood pressure and renal functional changes, we first determined the IC50 value of the effect of nitroprusside (SNP), a NO donor, on renal 20-HETE production in cortical microsomes. We then divided pregnant rats and age-matched virgin rats into a vehicle control group, an L-NAME treatment group (0.25 mg/ml in drinking water), and a group treated with L-NAME plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; CYP4A-selective inhibitor, 10 mg·kg–1·day–1 iv). After 4 days of treatment, we measured blood pressure, renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in each group. The addition of SNP (IC50 = 22 μM) decreased renal cortical 20-HETE production. In pregnant rats, L-NAME treatment led to significantly higher mean arterial pressure (MAP) and RVR, and lower RBF and GFR. Combined treatment with DDMS and L-NAME significantly attenuated the increases in MAP and RVR and the decrease in GFR, but not the reduction in RBF induced by L-NAME treatment. L-NAME and L-NAME plus DDMS had no significant impact on renal hemodynamics in virgin rats. In addition, chronic treatment with DDMS selectively inhibited cortical 20-HETE production without a significant effect on CYP4A expression in L-NAME-treated pregnant rats. In conclusion, NO effectively inhibits renal cortical microsomal 20-HETE production in female rats. In pregnant rats, the augmentation of renal 20-HETE production after NO inhibition is associated with increased MAP and RVR, whereas decreased GFR is negated by treatment of a selective and competitive CYP4A inhibitor. These results demonstrate that the interaction between renal 20-HETE and NO is important in the regulation of renal function and blood pressure in pregnant rats.
pregnancy; cytochrome P-450; arachidonic acid; eicosanoid; kidney; nitric oxide; hypertension
THE MAJOR CYTOCHROME P-450 (CYP)-derived eicosanoid in rat kidney, 20-hydroxyeicosatetraenoic acid (20-HETE), is formed primarily by CYP4A isoforms. The expression of CYP4A isoforms and production of 20-HETE in the small arterial vessels is well documented (12, 30, 34). In the microvessels, 20-HETE depolarizes vascular smooth muscle by inhibiting Ca2+-activated K+ channels and enhances the conductance of L-type Ca2+ channels (11, 40), thereby sensitizing vascular smooth muscle to constrictor stimuli and causing vasoconstriction (39). In the rat kidney, 20-HETE inhibits ion transport in the different segments of nephron. For example, in the renal tubules, 20-HETE inhibits Na+-K+-ATPase activity in a concentration-dependent manner (0.1 nM-1 μM) through phosphorylation of the -subunit of Na+-K+-ATPase by protein kinase C (25, 28). Escalante et al. (10) demonstrated the inhibitory effect of 20-HETE on sodium transport in the thick ascending limb of the loop of Henle (TALH). Interestingly, Wang and Lu (37), using a patch-clamp technique, demonstrated that 20-HETE blocks the 70-pS K+ channel in the apical membrane of TALH cells, thus limiting the amount of K+ available for transport via Na+-K+-2Cl– cotransporter and reducing the positive potential in the lumen, which is the main driving force for passive reabsorption of cations in the TALH. Because of these biological activities, 20-HETE has been linked to regulation of renal function and blood pressure in many animal models of hypertension (15, 20, 31).
Preeclampsia, which affects 5–10% of pregnancies in the U.S., is characterized by increased arterial blood pressure, generalized vasoconstriction, increased systemic resistance, widespread vascular endothelial damage, decreased fetal growth, and proteinuria (18). The exact mechanisms that mediate preeclampsia are still unknown. Several reports have suggested that nitric oxide (NO) may play an important role in its development (5, 19). Moreover, different investigators have demonstrated that chronic inhibition of NO synthesis in pregnant rats results in signs similar to those of preeclampsia (23, 38). Several studies have demonstrated that NO inhibits 20-HETE synthesis and interferes with 20-HETE vasoconstrictor activity in vivo (13, 27, 32). We previously demonstrated that NO binds differently to CYP4A1 and CYP4A3, the major renal CYP isoforms in female rats, and inhibits their catalytic activity (36). Moreover, the inhibition of NO production by N-nitro-L-arginine methyl ester (L-NAME) during late pregnancy in rats causes the augmentation of 20-HETE synthesis in renal microvessels (36). However, it is not known whether the upregulation of renal microvessel 20-HETE production affects renal function in L-NAME-treated pregnant rats.
In the present study, we examined the effect of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective CYP4A inhibitor, on various renal functional parameters, including mean arterial pressure (MAP), renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in pregnant rats treated with L-NAME during days 14-17 of gestation. Age-matched virgin rats served as controls. This study provides valuable information regarding the interaction between NO and 20-HETE in the regulation of renal function and blood pressure in pregnant rats.
MATERIALS AND METHODS
Materials. We obtained [1-14C]arachidonic acid (56 mCi/mmol) from DuPont-New England Nuclear (Boston, MA). All HPLC solvents and chemicals for buffer were obtained from Sigma (Milwaukee, WI). We purchased 20-HETE, epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) standard from Cayman Chemicals (Ann Arbor, MI).
Animals. All animals were purchased from Charles River Laboratories (Wilmington, MA). We conducted experiments in pregnant (timed pregnancy) and age-matched female Sprague-Dawley rats. All rats were maintained on a 12:12-h light-dark cycle and were housed two rats to a cage. All animal protocols were approved by the Institutional Animal Care and Use Committee and were in accordance with the protocols for animal use outlined in the Guide for the Care and Use of Laboratory Animals.
Protocol to evaluate the effect of sodium nitroprusside on renal microsomal 20-HETE production. We examined the stability of the NO donor sodium nitroprusside (SNP) with NO-sensitive litmus paper using Griess reagent [0.5 g of sulfanilamide plus 20 mg of N-(1-naphthyl)ethylenediamine dihydrochloride] dissolved in 10 ml of methanol (24). We preincubated SNP (15–56 μM, final concentration) with renal cortical microsomes (150 μg) isolated from female rats at 37°C for 20 min, then added [1-14C]arachidonic acid (0.4 μCi, 7 nmol), NADPH (1 mM), buffer (10 mM MgCl2 and 100 mM KH2PO4, pH 7.2) to the incubation mixtures in a final volume of 0.15 ml. Incubation was then carried out at 37°C for an additional 30 min. The reaction was terminated by acidification to pH 3.5–4.0 with 2 M formic acid. We extracted metabolites with ethyl acetate and determined 20-HETE production in the renal microsomes by HPLC as previously described (35). We also determined the IC50 value as previously described (33).
Protocol to evaluate the effect of DDMS on renal function in L-NAME-treated pregnant and virgin rats. We divided the pregnant rats (day 14 of gestation) and age-matched virgin rats into three groups (6 rats per group): 1) a vehicle-treated group (45% 2-hydroxypropyl--cyclodextrin), 2) an L-NAME treatment group; (0.25 mg/ml in drinking water), and 3) a group treated with L-NAME (0.25 mg/ml in drinking water) plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 10 mg·kg–1·day–1 iv). Each group was treated for 4 days. The dosages of L-NAME and DDMS were based on our previous studies and a literature search (4, 36). After treatment, we used these pregnant rats and age-matched female rats for a renal functional study. On day 18 of pregnancy, after finishing the functional study, we immediately removed their kidneys for renal cortical homogenate preparation to be used to assess renal 20-HETE production and CYP4A expression.
Renal functional measurements. We performed the renal functional study in pregnant rats and age-matched control female rats from the three treatment groups. Each rat was weighed before surgery. We anesthetized the rats with 2% isoflurane delivered by an anesthesia apparatus. Then, we placed one polyethylene cannula in the trachea (PE-205) to allow free breathing, one in the bladder (PE-240) to collect urine, one in the femoral artery (PE-50) for measuring and recording MAP with a pressure transducer, and one (PE-50) in the femoral vein for the infusion of agents. We then began infusing saline (3 ml/h iv) and a dose of 0.5 ml of FITC inulin (8 mg/ml in PBS, Sigma) was administered over 2 min as a priming dose. We performed a left laparotomy and placed a Transonic flow probe (Transonic System, Ithaca, NY) over the left renal artery to measure RBF. During the experiments, the rats’ body temperature was maintained at 37°C by a temperature controller (Cole Palmer Instrument) connected to a heating mat and a rectal temperature probe. After a volume of saline containing 6.2% BSA equal to 1.25% body wt had been infused, the intravenous infusion was switched to saline without BSA, but still with FITC inulin at 4 mg/ml. At least 45 min were allowed for equilibration following surgery before the beginning of the 30-min urine collections. Arterial blood (0.4 ml) was drawn from the femoral artery in the middle of each 30-min clearance period for measurement of GFR. An equal volume of normal saline was infused for volume replacement. MAP, RBF, and RVR were obtained from a computerized data collection and analysis system (EMKA Technologies, Falls Church, VA). We determined the concentration of FITC-inulin in plasma and urine using a GENios Plus fluorescent plate reader (Tecan, Research Triangle Park, NC) at 485-nm excitation and 538-nm emission. We used the concentration of FITC inulin in the plasma and urine to calculate GFR as described previously (29).
Isolation of renal cortical homogenates. After the renal functional study, we immediately isolated the kidneys from treated and control rats. Renal cortex from each rat was homogenized in 1 ml of 10 mmol/l potassium buffer (pH 7.7) containing 250 mmol/l sucrose, 1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonyl fluoride (PMSF), and 7.5 μl/ml protease inhibitor cocktail (Sigma). We centrifuged the homogenates for 15 min at 3,000 g and for 30 min at 11,000 g and collected supernatants and stored them at –80°C. The protocol for homogenate preparation was adapted from that of Hoagland et al. (14).
Arachidonic acid metabolism in renal cortical homogenates. We incubated renal cortical homogenates (1 mg) isolated from treated and control with [1-14C]arachidonic acid (0.2 μCi, 3.5 nmol) and NADPH (1 mmol/l, final concentration) in 1 ml potassium phosphate buffer (100 mmol/l, pH 7.4) containing 10 mmol/l MgCl2 for 15 min at 37°C. The reaction was terminated by acidification to pH 3.5–4.0 with 2 mol/l formic acid, after which arachidonic acid metabolites were extracted with ethyl acetate. We evaporated the ethyl acetate under nitrogen, resuspended the metabolites in 50 μl methanol, and injected them into the HPLC column. We performed reverse-phase HPLC on a 5-μm ODS-Hypersil column, 4.6 x 200 mm (Hewlett Packard, Palo Alto, CA) using a linear gradient of acetonitrile:water:acetic acid ranging from 50:50:0.1 to 100:0:0.1 at a flow rate of 1 ml/min for 30 min. The elution profile of the radioactive products was monitored by a flow detector (In/us System, Tampa, FL). We confirmed the identity of 20-HETE and DHETs with authentic standards. The activity of 20-HETE formation was estimated based on the specific activity of the added [1-14C]arachidonic acid and was expressed as pmoles per milligram of protein per minute as described previously (14, 35).
Western blot analysis. We separated renal cortical homogenates (10 μg) from treated and control rats by electrophoresis on a 10 x 20-cm, 8% SDS-polyacrylamide gel at 25 mA/gel at 4°C for 18–20 h. The proteins were transferred electrophoretically to an enhanced chemiluminescence (ECL) membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 10 mmol/l Tris·HCl, 0.1% Tween 20, and 150 mmol/l NaCl for 90 min, then washed three times with TBS. We incubated the membranes for 10 h with goat anti-rat CYP4A1 (1:2,000; Gentest, Woburn, MA) at room temperature, washed them several times with TBS, and incubated them again for 1 h with a 1:5,000 dilution of horseradish peroxidase-coupled rabbit anti-goat secondary antibody for CYP4A1. We developed the immunoblots using an ECL detection kit (Amersham, Arlington Heights, IL). To normalize the expression of CYP isoforms, we incubated renal cortical homogenates (10 μg) from treated and control rats with a 1:5,000 dilution of mouse anti-chicken -actin antibodies (Sigma, St. Louis, MO) for 10 h. The secondary antibody was horseradish peroxidase-coupled rabbit anti-mouse antibody (1:5,000). Immunoreactive -actin was detected as described earlier. We scanned the ECL films of Western blot analyses and performed densitometric analysis with Scion Image software using a gray color scale as a standard (SPSS 10.0).
Statistical analysis. Data are expressed as means ± SE. All data were analyzed by SPSS 10.0 computer software (SPSS, Chicago, IL). We used one-way ANOVA or Student’s unpaired two-tailed test for statistical analysis. Statistical significance was set at P < 0.05.
RESULTS
Effect of SNP on renal cortical microsomal 20-HETE production. To examine the stability of NO donor SNP, we incubated SNP (0.01 to 1 mM) at 37°C for varying times, then tested a drop of solution from each concentration with NO litmus paper prepared using filter paper that had been incubated with Griese reagent (24). As shown in Fig. 1A, SNP released NO (an orange spot shown by arrow bar) within 10–20 min of incubation. The litmus paper proved to be a convenient and useful method for qualitatively checking NO production and indicated that SNP stably released NO within 20 min at 37°C. To examine whether NO has an inhibitory effect on 20-HETE production in renal cortical microsomes, we incubated renal cortical microsomes isolated from female rats with SNP (15–56 μM) at 37°C for 20 min, then incubated them with arachidonic acid and NADHP and determined 20-HETE production by HPLC. As shown in Fig. 1B, the addition of SNP inhibited 20-HETE production in renal cortical microsomes in a concentration-dependent manner, with an IC50 of 22 μM.
Effects of DDMS on renal function of L-NAME-treated pregnant and control rats. The preceding study showed upregulation of renal vascular 20-HETE production after chronic treatment of pregnant rats with L-NAME (36). To assess the contribution of elevated 20-HETE production to renal hemodynamics after withdrawal of NO, we examined renal functional parameters in pregnant and age-matched virgin female rats treated with vehicle, L-NAME, or L-NAME plus DDMS. As shown in Fig. 2, MAP was significantly decreased on day 18 of gestation compared with that in control nonpregnant rats. L-NAME treatment from days 14 to 17 of gestation caused a significant elevation of blood pressure, from 97 ± 3 to 134 ± 4 mmHg. This elevation of MAP was attenuated by combined treatment with L-NAME and DDMS. RBF values were slightly increased in pregnant rats compared with virgin rats (6.1 ± 0.4 vs. 5.1 ± 0.4 ml/min). L-NAME treatment of pregnant rats significantly decreased RBF from 6.1 ± 0.4 to 3 ± 0.3 ml/min, whereas L-NAME plus DDMS treatment did not significantly alter RBF in L-NAME-treated pregnant rats. RVR values in pregnant rats were significantly lower than those in nonpregnant control rats (15.3 ± 1.7 vs. 21.3 ± 1.6 mmHg·ml–1·min–1, P < 0.05). L-NAME treatment of pregnant rats also caused a significant increase in RVR, from 15.3 ± 1.7 to 45.3 ± 4.0 mmHg·ml–1·min–1. Moreover, the elevation of RVR in response to L-NAME was attenuated by treatment with L-NAME plus DDMS. As shown in Fig. 3, the GFR values in pregnant rats were significantly higher than those in nonpregnant control rats (1.6 ± 0.1 vs. 1.1 ± 0.04 ml/min, P < 0.05). L-NAME treatment markedly decreased GFR in pregnant rats to the levels in nonpregnant rats. This reduction of GFR by L-NAME treatment was attenuated by treatment with L-NAME plus DDMS (Fig. 3). In contrast, the MAP, RBF, RVR, and GFR in L-NAME-treated virgin rats did not significantly differ from control rats (Figs. 2 and 3). Similarly, combined treatment with L-NAME and DDMS had no impact on the changes in MAP, RBF, RVR, and GFR in virgin rats (Figs. 2 and 3).
Effects of DDMS on renal cortical 20-HETE production of L-NAME-treated rats. To study the selectivity of the effect of DDMS on renal 20-HETE synthesis, we used HPLC to examine renal cortical arachidonic acid metabolism in pregnant rats treated with L-NAME and L-NAME plus DDMS. As shown in Fig. 4A, incubation of renal cortical homogenates isolated from L-NAME treatment with [14C]arachidonic acid and NADPH produced DHETs and 20-HETE. After treatment with DDMS plus L-NAME, the conversion of arachidonic acid to 20-HETE (-hydroxylase activity) decreased by 44%. DHETs production was not affected compared with L-NAME treatment (Fig. 4A). The -hydroxylase activity in the L-NAME treatment group was 49 ± 4 pmol·min–1·mg protein–1. Treatment with DDMS plus L-NAME caused a 44% reduction in this activity to 26 ± 3 pmol·min–1·mg protein–1 (n = 4, P < 0.05). The selective inhibition of 20-HETE by DDMS treatment was also observed in L-NAME plus DDMS-treated virgin rats (data not shown). To examine the effect of DDMS treatment on CYP4A expression, we conducted Western blot analysis for CYP4A in renal cortical homogenates isolated from pregnant rats treated with L-NAME and DDMS plus L-NAME. A representative Western blot of CYP4A isoforms after two treatments is shown in Fig. 4B. Renal cortical homogenate CYP4A protein levels were similar in rats treated with L-NAME and DDMS plus L-NAME.
DISCUSSION
Normal pregnancy in rats is associated with increases in GFR and RBF and reductions in RVR and MAP (1, 17). Many different investigators have suggested that NO plays an important role in the regulation of renal function and blood pressure in pregnant rats (18, 19). NO causes the relaxation of smooth muscle in blood vessels, and the mechanisms for the action of NO on blood pressure regulation during pregnancy may be attributable to the upregulation of NO synthesis in blood vessels. It is also possible that pregnancy-induced changes in hormonal background contribute to the upregulation of NO production. Supporting this hypothesis, a prior study by Chen et al. (7) showed that estrogen stimulates NO synthesis through endothelial NO synthase (eNOS). Another study by Binko and Majewski (6) showed that estrogen increases the expression of inducible NO synthase (iNOS) in the vascular smooth muscle of blood vessels. In addition, Conrad et al. (8) demonstrated that the binding of NO to hemoglobin is detected only in the blood of pregnant, not nonpregnant, rats and Alexander et al. (1) demonstrated that increased NO production in pregnant rats is associated with upregulation of neuronal NO synthase (nNOS) and iNOS in the kidneys. These findings support the notion that upregulation of the NO system may be important in regulating vascular tone and blood pressure during pregnancy. To investigate the role of NO on the regulation of functional changes during pregnancy, Kassab et al. (16) used L-NAME, a competitive inhibitor of NO synthase, to study the consequence of NO inhibition on blood pressure and systemic and renal hemodynamic changes in pregnant rats. In this study, L-NAME-induced hypertension in pregnant rats is associated with increased RVR and decreased RBF (16). However, the mechanisms whereby L-NAME causes these changes in renal hemodynamics are still not very clear.
One possibility is that chronic blockade of NO by L-NAME in pregnant rats results in the amplification of renal vasoconstrictor factors that cause the changes in renal function. In a previous study, we demonstrated a marked increase in renal vascular 20-HETE production after L-NAME treatment of pregnant rats (36). Because 20-HETE is a vasoconstrictor of renal arterioles, we hypothesize that this increased renal vascular 20-HETE synthesis after chronic NO inhibition may contribute to the alteration of renal function in L-NAME-treated pregnant rats. The essential findings of the present study, that selective inhibition of renal 20-HETE production in pregnant rats attenuates the changes in renal hemodynamics caused by L-NAME treatment, are in accord with that hypothesis. This conclusion is based on the observation that L-NAME-induced renal functional changes such as increased MAP, increased RVR, and decreased GFR are attenuated by treatment with DDMS, a selective inhibitor of 20-HETE production. Although we observed attenuation of the changes of renal hemodynamics by DDMS treatment in L-NAME-pregnant rats, this treatment cannot totally reverse the changes in renal hemodynamics, returning them back to the levels in control pregnant rats (Figs. 2 and 3), perhaps because of increases in the sensitivity of vasoconstriction systems other than 20-HETE after NO blockade. Molnar and Hertelendy (22) demonstrated that in rats at late pregnancy, acute treatment with L-NAME enhances the sensitivity of the pressor responses to angiotensin II, norepinephrine, and arginine vasopressin (22). Edwards et al. (9) showed that L-NAME treatment during mid to late pregnancy causes elevation in the level of plasma endothelian-1. However, further investigation is needed to determine whether these vasoconstriction systems are involved in the regulation of renal hemodynamics after chronic NO blockade.
Besides its action on vascular relaxation, NO inhibits CYP enzyme systems (21, 31). The mechanisms for the inhibition of CYP enzymes by NO are thought to be reversible binding between NO and the heme moiety of CYP isoforms and irreversible modification of CYP isoforms by NO (21). In this regard, Sun et al. (32) showed that the addition of NO donors to recombinant CYP4A2 leads to the binding of NO to the heme moiety of CYP4A2 (an increases in the visible light absorbance at 440 nm). Minamiyama et al. (21) showed that NO can irreversibly modify the cysteine residues of CYP proteins. More recently, we demonstrated that NO binds to the heme moiety of CYP4A1 and CYP4A3 isoforms, the major enzymes for renal 20-HETE production in female rats, and that the NO donor SNP inhibits both CYP4A1 and CYP4A3-catalyzed 20-HETE production (36). These results are consistent with the results shown in Fig. 1, that SNP constantly releases NO and that SNP inhibits renal 20-HETE production in female rats. These results and those from our previous study (36) provide solid biochemical evidence of the interaction between NO and CYP4A enzymes and this interaction results in the inhibition of 20-HETE production in the kidneys.
Several investigators demonstrated the functional implications of interaction between NO and 20-HETE in vivo. For example, Magdalena et al. (2) found that acute administration of DDMS attenuated the NO-mediated vasodilatory response in rat renal arterioles and that a reduction in MAP and RVR was mediated by NO donor. Another report by the same group demonstrated that inhibition of 20-HETE production by DDMS treatment contributes to the cerebral vasodilation response to NO in rats (3). In addition, Oyekan and McGiff (26) showed that acute inhibition of 20-HETE by 12,12-dibromododec-enoic acid, another selective inhibitor of 20-HETE production (33), attenuated acute L-NAME-induced reductions in RBF and GFR, as well as increases in MAP and RVR in male rats. More recently, Hercule et al. (13) demonstrated that the treatment of antisense oligodeoxynucleotides for CYP4A isoforms attenuates both the reduction in RBF and the increase in RVR induced by L-NAME treatment. Because we did not observe the significant renal hemodynamic changes in L-NAME and L-NAME plus DDMS-treated virgin rats (Figs. 2 and 3), the interaction between NO and CYP4A isoforms may be a unique and important mechanism in the regulation of renal function during pregnancy. In pregnant rats, the overproduction of NO in the kidneys (1) can lead to binding of NO to CYP4A isoforms in renal arterioles and mask the vasoconstrictive effect of 20-HETE during normal pregnancy. When chronic L-NAME treatment blocks NO production, NO no longer keeps the 20-HETE system under control resulting in the elevation of renal vascular 20-HETE production (36) and the amplification of renal vasoconstriction mechanisms that cause increased RVR and MAP (Fig. 2). More importantly, we showed that the increases in RVR and MAP induced in L-NAME-treated pregnant rats were attenuated by the inhibition of renal 20-HETE synthesis (Fig. 2). Taken together, the present study solidly demonstrates that the interaction between NO and 20-HETE has many functional implications with respect to the regulation of blood pressure and renal function in pregnant rats.
It is well recognized that 20-HETE is formed endogenously in various tissues and exerts potent biological effects on cellular functions. Studies of its role on cellular levels are impeded by the difficulty in selectively targeting its synthesis and effects. This difficulty arises from that fact that the commonly used CYP inhibitors such as 1-aminobenzotriazole and 1-octadecynoic acid do not selectively inhibit 20-HETE production (31). We previously characterized several selective inhibitors of 20-HETE production in renal microsomes (33). Among them, DDMS was found to be highly specific for 20-HETE production, with an IC50 value about 2 μM, whereas its IC50 value for EETs formation is 60 μM. In the present study, we used DDMS to block the arachidonic acid -hydroxylase pathway and found that DDMS specifically blocked 20-HETE production without significantly affecting DHETs formation in pregnant rats (Fig. 4A). In addition, chronic DDMS treatment did not affect the renal expression of CYP4A isoforms in L-NAME-treated pregnant rats (Fig. 4B), which suggests that DDMS is a competitive inhibitor. Similar results for the competitive inhibition of 20-HETE by chronic DDMS treatment have been reported in the literature (4). Thus these data demonstrate that the selective inhibition of 20-HETE production by DDMS in vivo will be a very useful tool for elucidating the effect of 20-HETE in mediating physiological functions.
In summary, we presented evidence that chronic inhibition of NO production by L-NAME amplifies a vasoconstrictor system operating through 20-HETE that contributes to the changes in renal hemodynamics following suppression of NO production in pregnant rats. This conclusion is supported by the chronic effect of DDMS, a selective inhibitor of 20-HETE production, which inhibits the renal response to L-NAME-treated pregnant rats. This study demonstrates that activation of the renal 20-HETE pathway after removal of NO in the kidneys during pregnancy affects the regulation of renal function and blood pressure and that interaction between NO and 20-HETE may have important functional implications during pregnancy.
GRANTS
This study was supported by National Institutes of Health (NIH) Grant R01-HL-70887 to M.-H. Wang and by NIH Grant DK-38226 and a grant from Robert A. Welch Foundation to J. R. Falck.
ACKNOWLEDGMENTS
The authors thank Dr. D. M. Pollock for providing the protocol for measuring GFR. The authors also thank J. Cole for editorial assistance.
FOOTNOTES
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.
H. Huang and Y. Zhou contributed equally to the development of this research study.
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