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Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels

Center for Cardiovascular Diseases, College of Pharmacy and Health Sciences, Texas Southern University, Houston, Texas 77004; and ²Department of Pharmacology, New York Medical College, Valhalla, New York 10595

Submitted 27 January 2003 ; accepted in final form 15 July 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Nitric oxide (NO) is an inhibitor of hemoproteins including cytochrome P-450 enzymes. This study tested the hypothesis that NO inhibits cytochrome P-450 epoxygenase-dependent vascular responses in kidneys. In rat renal pressurized microvessels, arachidonic acid (AA, 0.03–1 µM) or bradykinin (BK, 0.1–3 µM) elicited NO- and prostanoid-independent vasodilation. Miconazole (1.5 µM) or 6-(2-propargyloxyphenyl)hexanoic acid (30 µM), both of which are inhibitors of epoxygenase enzymes, or the fixing of epoxide levels with 11,12-epoxyeicosatrienoic acid (11,12-EET; 1 and 3 µM) inhibited these responses. Apamin (1 µM), which is a large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel inhibitor, or 18-glycyrrhetinic acid (30 µM), which is an inhibitor of myoendothelial gap junctional electromechanical coupling, also inhibited these responses. NO donors spermine NONOate (1 and 3 µM) or sodium nitroprusside (0.3 and 3 µM) but not 8-bromo-cGMP (100 µM), which is an analog of cGMP (the second messenger of NO), blunted the dilation produced by AA or BK in a reversible manner without affecting that produced by hydralazine. However, the non-NO donor hydralazine did not affect the dilatory effect of AA or BK. Spermine NONOate did not affect the dilation produced by 11,12-EET, NS-1619 (a BK_Ca channel opener), or cromakalim (an ATP-sensitive K⁺ channel opener). AA and BK stimulated EET production, whereas hydralazine had no effect. On the other hand, spermine NONOate (3 µM) attenuated basal (19 ± 7%; P < 0.05) and AA stimulation (1 µM, 29 ± 9%; P < 0.05) of renal preglomerular vascular production of all regioisomeric EETs: 5,6-; 8,9-; 11,12-; and 14,15-EET. These results suggest that NO directly and reversibly inhibits epoxygenase-dependent dilation of rat renal microvessels without affecting the actions of epoxides on K⁺ channels.

epoxides; cytochrome P-450; vasodilation

This study was designed to evaluate the effects of NO on epoxygenase activity and epoxygenase-dependent vascular effects in rat renal preglomerular vessels. Our data provide evidence that NO inhibits basal and AA-stimulated production of all of the regioisomers of epoxides and reversibly inhibits epoxygenase-dependent dilation of the rat renal preglomerular vasculature without directly affecting the actions of epoxides themselves.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
These studies were performed using male Sprague-Dawley rats (12–13 wk; body wt, 343 ± 8 g) obtained from Harlan Sprague Dawley (Houston, TX). The animals were maintained on a standard rat food (Purina Chow) and were allowed ad libitum access to water and food until the beginning of the experiments. The study protocol was approved by the Animal Care and Use Committee of Texas Southern University.

A standard in vitro pressurized arteriole preparation was used to study responses of the renal preglomerular (arcuate and interlobar) vessels. Preglomerular vessels [intraluminal diameter (ID), 140–210 µm; length, 1 mm] were carefully removed, cleaned with the aid of a dissecting microscope, and placed in an organ chamber. Each end of the microvessel was cannulated with a glass micropipette and secured with 10-0 ophthalmic suture. The organ chamber was placed on the stage of an inverted microscope. Attached to the microscope were a video camera, a video monitor, and a calibrated video caliper. The organ chamber was connected to a rotary pump that continuously circulated oxygenated Krebs-Henseleit buffer that contained (in mM) 118.3 NaCl, 4.7 KCl, 1.2 CaCl₂, 1.2 MgS0₄, 25.0 NaHC0₃, 1.2 KH₂P0₄, and 5.5 glucose. Solutions were aerated with 95% O₂-5% CO₂ and maintained at 37°C, pH 7.4. An image of the microvessel was displayed on the video monitor, and ID was measured by online image processing using a video micrometer. The resolution of the system allowed measurement of very small (5 µm) changes in vessel diameter. Microvessels were allowed to equilibrate for 45–60 min at a hydrostatic distending pressure of 80 mmHg. Phenylephrine (PE, 0.3 µM) was added to the bath to test the constriction capacity of the vessel; 70–80% constriction was deemed adequate for these studies. Cumulative concentration-response relationships were established for AA (0.03–1 µM), BK (0.1–3 µM), or hydralazine (negative control, 0.5–15 nM) in preconstricted vessels by adding the drugs directly to the organ bath. Vasodilator effects of the agonists on the renal preglomerular vessels were measured 3 min after the extraluminal applications. Responses to agonists were determined in the same vessel with a 15- to 20-min washout period between applications.

To isolate epoxide-mediated dilation, microvessels were routinely incubated in Krebs buffer that contained indomethacin (10 µM) to inhibit the generation of prostanoids. Unless otherwise stated, N-nitro-L-arginine methyl ester (L-NAME, 100 µM), an inhibitor of NOS, was also routinely added to eliminate the effects of endogenously generated NO. The role of CYP-dependent epoxygenase in AA- or BK-evoked vasodilation of the renal microvessels was evaluated using two mechanistically distinct inhibitors, namely, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH, 30 µM, n = 5), which is a selective substrate inhibitor (IC₅₀ of 9 µM for epoxygenase; Ref. 38), and miconazole (1.5 µM, n = 6), which acts on the heme moiety of CYP (38). To evaluate the role of K⁺ channels, responses to agonists were determined in the presence of apamin (1 µM), which is a large-conductance Ca²⁺-activated (BK_Ca) channel antagonist. Because miconazole affects K⁺ channels, the specificity of the effect of miconazole was evaluated by testing its effects on the renal microvascular response to 11,12-epoxide (1 and 10 µM, n = 4 or 5). The role of gap junctions in epoxide-mediated vasodilation was evaluated in the presence of 18-glycyrrhetinic acid (AGRA, 30 µM, n = 4), which is an inhibitor of myoendothelial gap junctional electromechanical coupling (32). The concentrations of the inhibitors used in these studies were based on their IC₅₀ values or published literature from our laboratory and others (25, 27, 32, 38). Because the inhibitors used in these studies did not affect the reactivity of the vessels, a paired protocol was used whereby the experiments were performed as sequential measurements before and after application of inhibitors, which allowed the same vessels to be studied. When we evaluated the effects of the inhibitors on agonist-induced dilation, an inhibitor-tissue contact time of 30 min was allowed before concentration-response curves were established. A vessel was usually used for not more than two inhibitors as long as there was a full recovery to the effects of AA when the first inhibitor was washed off and the tissue was bathed with fresh buffer (without the inhibitor). To investigate the role of NO, responses to AA or BK were determined in the presence of the NO donors spermine NONOate (1 and 3 µM, n = 5) or sodium nitroprusside (SNP, 0.3 and 3 µM, n = 5) or 8-bromo-cGMP (100 µM, n = 5), which is a cell-permeable analog of cGMP, the second messenger of NO. To determine whether a non-NO donor vasodilating substance is associated with the observed inhibition of the response, dilation induced by AA was also evaluated in the presence of hydralazine (1.5 and 5 nM). In experiments where L-NAME was used, the microvessel required PE (0.1 µM) to produce similar tone as vessels preconstricted with PE (0.3 µM) alone. In experiments to evaluate the effects of NO donors on dilation induced by other vasodilators, e.g., 11,12-epoxide, NS-1619, etc., the concentration of PE was increased sufficiently to override the dilation that was produced and to elicit comparable constriction of preglomerular vessels as vessels that were not treated with NO donors. In some experiments, the reversibility of the effects of NO on epoxide-mediated dilation was determined by measurement of agonist-induced dilation after replacement of the buffer that contained spermine NONOate or SNP with fresh Krebs buffer. In additional experiments, the specificity of the effect of NO on epoxygenase was determined by evaluating the effects of spermine NONOate (1 µM) on the dilator responses to 11,12-epoxide (1, 3, and 10 µM), the BK_Ca channel agonist (8) NS-1619 (10 and 20 µM), or the ATP-sensitive K⁺ (K_ATP) channel-selective agonist cromakalim (3, 10, and 30 µM).

Measurement of vascular production of epoxides. Preglomerular vessels were microdissected and incubated in vials that contained 1 ml of oxygenated Krebs buffer to which NADPH (1 mM final concentration) and indomethacin (10 µM final concentration) were added. Vessels harvested from both kidneys of the same rat were homogenized in PBS at 4°C. Aliquots (50 µl) were saved for protein determination (via Bradford assay) using BSA as standard. Each 200-µl aliquot was transferred into four vials that contained buffer to which either vehicle, spermine NONOate (3 µM final concentration), hydralazine (5 nM), BK (3 µM), AA (1 µM), or AA (1 µM) and spermine NONOate (3 µM final concentration) were added. This protocol allowed us to evaluate the effects of NO on basal and AA-stimulated production of epoxides. The samples were incubated for 1 h at 37°C in an atmosphere of 95% O₂-5% CO₂. At the end of the incubation period, acetic acid (50 µl) was added to stop the reaction. Thereafter, both media and vessels were extracted with ethyl acetate, and epoxides present in the extract were purified and quantitated by a modification of the negative chemical ionization gas chromatography-mass spectroscopy (GC/MS) method that we have previously described (25). In some experiments (n = 3), "blank" controls (buffer without microvessels) were added and subjected to the same procedure.

Because all four of the EET isomers (5,6-; 8,9-; 11,12-; and 14,15-EET) show up as one peak and there are too many impurity peaks interfering with the direct detection of EETs by GC/MS, an improved method to quantitate the EETs was used that involves converting EETs to dihydroxyeicosatrienoic acid (DHET) by acid hydrolysis. To each 1-ml sample, 3 ng of the deuterium-labeled internal standard (1 ng each of 14,15-; 11,12-; 8,9-; and 5,6-EET-d₈) were added. Samples were then acidified to pH 4 with 10% acetic acid. Ethyl acetate (1 ml) was added, the sample was vortexed and centrifuged, and the supernatant was collected. This procedure was performed twice. The extracts were pooled, dried under a gentle nitrogen flow, and then dissolved in 100 µl of 0.5 µM sulfuric acid. After 30 min at 60°C, the samples were neutralized and adjusted to pH 4 with 1 M potassium hydroxide. Ethyl acetate was again used to extract the hydrolyzed products of the EETs, which were then dried and dissolved in methanol (50 µl) for HPLC separation. HPLC was performed on an HP1050 instrument with a Beckman ODS column (25 cm x 4.6 mm, 5 µm) using the same gradient for 20-HETE (25). The collected HPLC fraction that corresponded to the four regioisomers of DHETs were dried, derivatized, and subjected to GC/MS detection by monitoring ions at mass-to-charge ratios (m/z) of 481 and 489, which represent the derivatives of the native and d₈-labeled DHETs, respectively. The derivatization procedure was the same as we previously reported for 20-HETE (25). Authentic standards were used to identify the specific EETs. The 11,12-; 8,9-; and 5,6-DHETs showed up with the same retention time on GC/MS and were quantitated together, whereas the 14,15-DHET appeared as a separate peak. The EET concentrations were calculated from the peak area ratio of m/z values of 481 and 489 according to the standard curve of DHET vs. DHET-d₈.

Solutions and drugs. AA (a sodium salt) was obtained from Nuchek (Elysian, MN). L-NAME, miconazole, SNP, indomethacin, hydralazine, AGRA, 8-bromo-cGMP, apamin, NS-1619, and cromakalim were from Sigma Chemical (St. Louis, MO). 11,12-EET, deuterated EETs, PPOH, and spermine NONOate were from Cayman Chemical (Ann Arbor, MI). All solutions and vasoactive agents were prepared fresh or diluted from stock solutions on the day of the experiment. AA, SNP, and L-NAME were dissolved in normal saline (0.9% NaCl). Miconazole, PPOH, indomethacin, or AGRA was initially dissolved in 100% ethanol to provide a stock solution of 10–100 mM and was subsequently added to Krebs buffer to provide the desired concentration of ethanol (<2%) that did not affect AA- or hydralazine-induced vasodilation.

Statistical analysis. Data are presented as means ± SE. Changes in ID of the vessels in response to the vasodilators are expressed as absolute changes () relative to the preconstricted tone. Prism 3.0 software (GraphPad) was used for statistical analyses. All concentration-response curves were evaluated for differences using two-way repeated-measures ANOVA followed by Newman-Keuls test. In some cases, Student's t-test for paired data was used to determine significance. In all cases, differences with P 0.05 were considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Agonist-induced vascular responses. Baseline diameters of the isolated pressurized preglomerular vessels in these experiments ranged between 140 and 210 µm. Application of pharmacological inhibitors of metabolic pathways, i.e., indomethacin (10 µM), miconazole (1.5 µM), PPOH (30 µM), or L-NAME (100 µM), did not impair the ability of the vessels to dilate: the dilatory actions of hydralazine were not altered after any of these treatments. On its own, L-NAME (100 µM) produced a 44 ± 8% increase in vascular tone of the microvessel; thus PE (0.1 µM) was always added to produce the required 70–80% increase in basal tone. Figure 1 presents data that describe changes in vessel ID in response to AA, BK, or hydralazine. In the indomethacin-treated vessel constricted with PE and L-NAME, AA (0.03–1 µM) elicited a dose-dependent increase in ID from a preconstricted diameter of 78 ± 7 µm to a value that was 42 ± 6% of the constricted tone at the highest concentration employed. There was no tachyphylaxis to the effects of AA. Similarly, BK (0.1–3 µM) and hydralazine (0.5–15 nM) produced dose-dependent vasodilations that reached values that were 53 ± 6 and 68 ± 5% of the constricted tone at the highest concentrations employed. In the absence of L-NAME, AA-induced vasodilation was not different in the indomethacin-treated preglomerular vessel preconstricted with PE (0.3 µM, n = 5) alone (Table 1). For example, at the highest concentration of AA (1 µM), the increase in ID was 50 ± 5% of the 0.3-µM PE-preconstricted tone. This value is not much different from that produced in vessels preconstricted with PE and L-NAME, 43 ± 6%. Similar to AA, hydralazine-induced dilation was not different in the absence or presence of L-NAME (Table 1). However, the BK-induced increase in ID was less (35 ± 4%; P < 0.05) in vessels treated with L-NAME and PE compared with those treated with PE alone (Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effects of miconazole (MCNZ, 1.5 µM) or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH, 30 µM) on changes in vessel diameter of pressurized renal microvessels in response to extraluminal application of arachidonic acid (AA, A), bradykinin (BK, B), or hydralazine (C). Vessels were incubated in buffer that contained indomethacin (10 µM) and were preconstricted with phenylephrine (PE, 0.1 µM) and N-nitro-L-Arginine methyl ester (L-NAME, 100 µM). Inhibitors were added to the buffer for at least 30 min before the effects of the agonists were evaluated. Control responses are those obtained in vehicle-treated vessels before the addition of miconazole or PPOH. n, Number of kidneys from different rats. *P < 0.05 vs. control.

Characterization of dilator responses of renal microvessels: Roles of epoxides, K⁺ channels, and gap junctions. The results of experiments directed at determining the contribution of epoxides to dilator effects of AA and BK are also presented in Fig. 1. In the indomethacin-treated PE- and L-NAME-constricted preglomerular vessels, miconazole (1.5 µM), which is an inhibitor of CYP-dependent epoxygenase enzyme, markedly blunted the vasodilator responses to AA (64 ± 7%; P < 0.05; n = 6) and BK (89 ± 9%; P < 0.05; n = 6), as did PPOH (30 µM, n = 5), which inhibited AA and BK responses by 82 ± 6 and 92 ± 4%, respectively (P < 0.05; n = 5). However, neither miconazole nor PPOH had an effect on vasodilation elicited by hydralazine (n = 4). Miconazole also did not affect the dilator responses to 11,12-epoxide; if anything, miconazole tended to enhance its effects (data not shown). For example, 11,12-epoxide at 1, 3, and 10 µM increased the ID of the vehicle-treated renal microvessel (n = 4) by 14 ± 4, 20 ± 11, and 10 ± 21 µm, respectively. The corresponding responses in miconazoletreated vessels were 23 ± 4, 33 ± 5, and 39 ± 6 µm, respectively (n = 4).

Fixing the CYP products to saturating levels is a method that can be used to investigate their contributions to the reactivity of tissues (10). Figure 2A summarizes data that demonstrate the effects of fixing the level of 11,12-epoxide on dilation elicited by AA and hydralazine. At 1 and 10 µM, 11,12-epoxide blunted AA-induced vasodilation by 38 ± 8% (P < 0.05; n = 5) and 78 ± 8% (P < 0.05; n = 4), respectively. Similar levels of inhibition were observed when BK responses were evaluated (data not shown). By contrast, dilation elicited by hydralazine (n = 4 or 5) was not affected (Fig. 2B).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Effects of 11,12-epoxyeicosatrienoic acid (11,12-EET; 1 and 10 µM) added for 5 min before evaluation of the dilator effects of AA (A) or hydralazine (B) in the indomethacin- and L-NAME-treated PE-preconstricted microvessels. n, Number of vessels isolated from different kidneys from different rats. *P < 0.05 vs. control.

Because K⁺ channels have been implicated in the dilator effects of epoxides in various blood vessels (5, 12, 13, 30), we evaluated the effects of the BK_Ca channel inhibitor apamin on AA-induced vasodilation. Table 1 demonstrates that apamin (1 µM) markedly inhibited the increase in ID by AA (78 ± 8%; P < 0.05), BK (80 ± 8%; P < 0.05), and hydralazine (69 ± 11%; P < 0.05). Hyperpolarization-mediated responses were shown to involve myoendothelial gap junctions (31); therefore, we also evaluated the contribution of gap junctions to epoxide-mediated vasodilation. AGRA (30 µM), which is a gap junction inhibitor (32), blunted AA- and BK-stimulated increases in ID of the renal microvessels by 50 ± 5 and 65 ± 7% (P < 0.05), respectively, and that produced by hydralazine (Fig. 3).

View larger version (12K): [in this window] [in a new window]   Fig. 3. Changes in vessel diameter produced by AA (A), BK (B), or hydralazine (C) before (control) or after the addition of 18-glycyrrhetinic acid (AGRA) to Krebs buffer that bathed the renal microvessel. *P < 0.05 vs. control.

Effects of NO and 8-bromo-cGMP on AA- and BK-induced vasodilations. Figure 4 summarizes data that describe the effects of treating isolated pressurized preglomerular vessels with the NO donor spermine NONOate on dilator responses to AA, BK, or hydralazine. On its own, spermine NONOate at 1, 3, and 10 µM (n = 5) increased the ID of the indomethacin-pretreated PE- and L-NAME-constricted preglomerular vessel by 14 ± 3, 30 ± 6, and 73 ± 9 µm, respectively, from a basal diameter of 70 ± 10 µm. However, spermine NONOate at 1 and 3 µM attenuated AA- and BK-induced vasodilation in a dose-related manner by 40 ± 5 and 82 ± 11%, respectively (P < 0.05; n = 5) (Fig. 4A). Spermine NONOate also blunted the vasodilation produced by BK by 55 ± 9 and 87 ± 14%, respectively (P < 0.05; n = 5) but did not affect the vasodilation produced by hydralazine (n = 5; Fig. 4). The effects produced by spermine NONOate are reversible: vessels (n = 5) challenged with AA 20 min after washout of spermine NONOate (3 µM) responded to the same extent (92–98%) as before treatment with spermine NONOate (data not shown). Unlike the inhibition produced by spermine NONOate, 8-bromo-cGMP (100 µM), which is a cell-permeable analog of cGMP, the second messenger of NO, did not affect the dilation produced by AA or BK (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Changes in vessel diameter produced by AA (A), BK (B), or hydralazine (C) before (control) or 3–5 min after the addition of spermine NONOate (NONOate) to Krebs buffer that bathed the renal microvessel. 8-BrcGMP, 8-bromo-cGMP. *P < 0.05 vs. control.

Figure 5 presents additional evidence that NO affects dilation produced by AA or BK. Thus SNP (another NO donor) blunted the vasodilation produced by AA (n = 5) or BK (n = 5) without affecting that produced by hydralazine (n = 5). The inhibition by SNP of AA-induced vasodilation amounted to 37 ± 12 and 76 ± 7% at concentrations of 0.3 and 3 µM, respectively (P < 0.05), whereas the inhibition by SNP of BK-induced vasodilation was 41 ± 6 and 63 ± 7% (P < 0.05), respectively. Similar to the observation made of spermine NONOate, the effects produced by 3 µMSNP were fully reversed after SNP was washed out and the responses to AA were retested (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Dilator effect of AA (A), BK (B), or hydralazine (C) in the absence (control) of or 3–5 min after the addition of sodium nitroprusside (SNP). n, Number of vessels isolated from different kidneys from different rats. *P < 0.05.

In additional experiments to evaluate the specificity of the effects of NO on epoxide-mediated vasodilation, responses to AA or BK were not different in vessels (n = 4) treated with 1.5 and 5 nM hydralazine (a non-NO donor) compared with those treated with vehicle (Table 2).

Effects of 11,12-EET and activators of K⁺ as affected by NO. NO can activate K⁺ channels (24, 37) and thereby modulate the actions of agonists that activate these channels. The results of experiments to evaluate whether the inhibition by NO of AA-induced dilation of the preglomerular vessel is at the level of epoxygenase enzyme or beyond it at the K⁺ channel is shown in Fig. 6. Spermine NONOate, at a concentration that blunted the effects of AA and BK (1 µM; see Fig. 4), did not affect the dilator responses of 11,12-epoxide (n = 4; Fig. 6A). Instead, responses to 11,12-epoxide tended to increase in the presence of spermine NONOate. Similarly, spermine NONOate did not inhibit the dilation produced by NS-1619, which is a BK_Ca channel activator (n = 4; Fig. 6B), nor the dilation produced by cromakalim, which is a K_ATP channel activator (n = 3; Fig. 6C).

View larger version (19K): [in this window] [in a new window]   Fig. 6. Effects of spermine NONOate on changes in vessel diameter in response to extraluminal application (for 3 min) of 11,12-EET (A), NS-1619 (B), or cromakalim (C). Spermine NONOate was added to the buffer for 3–5 min before responses to the other agents were tested. n, Number of kidneys (vessels) from different rats. *P < 0.05 vs. control.

Epoxide production as affected by NO. Figure 7 summarizes data that describe the effects of treating microdissected preglomerular vessels (n = 4–6) and measuring the production of epoxides under basal conditions with BK, hydralazine (a non-NO donor), or spermine NONOate (a NO donor) in the absence or presence of AA. Basal production of EETs was 622 ± 148 pg/mg protein. AA (1 µM) and BK (3 µM) stimulated epoxide production in preglomerular vessels by 53 ± 11% (P < 0.05) and 34 ± 7% (P < 0.05), respectively, whereas hydralazine (5 nM) did not alter EET production. However, spermine NONOate (3 µM) reduced basal EET production in preglomerular vessels by 19 ± 7% (P < 0.05), and AA (1 µM) stimulated epoxide production by 29 ± 9% (P < 0.05). There was no quantitative or qualitative difference in the effects of spermine NONOate on any of the regioisomers of EET, as inhibition of the product identified as 14,15-EET or the mixture of 5,6-; 8,9-; and 11,12-EET was similar under basal or AA-stimulated conditions.

View larger version (22K): [in this window] [in a new window]   Fig. 7. A typical gas chromatography-mass spectroscopy (GC/MS) tracing (A) that illustrates the peaks of derivatized samples of 5,6-; 8,9-; and 11,12-dihydroxyeicosatrienoic acid (DHET; retention time, 4.30–4.35) or 14,15-DHET (retention time, 4.35–4.39) when the mass-to-charge ratios were monitored for ions of 481 and 489, which corresponds to native DHETs or deuterium (d8)-labeled DHETs, respectively. Spectra reveal the DHETs in buffer without microvessels (blank, a), buffer containing microvessels alone (basal, b) and microvessels challenged with 1 µM AA alone (d) or in the presence of 3 µM spermine NONOate (AA + spermine NONOate, c). Tracing is representative of at least 24 samples. Concentrations of 5,6-; 8,9-; 11,12-; and 14,15-epoxide (B) in plain buffer (blank) or from the media of renal microvessels without (basal) or following addition to the buffer (for 30 min) of 3 µM spermine NONOate, 3 µM BK (BKN), 5 nM hydralazine, or 1 µM AA alone or with spermine NONOate (AA + NONOate). In all cases, media collected were subjected to HPLC fractionation and GC/MS analysis as described in MATERIALS AND METHODS. *P < 0.05 vs. basal; @P < 0.05 vs. AA.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This study demonstrates that dilation of renal preglomerular vessels in response to AA and BK has a component that 1) is predominantly mediated by an epoxygenase-derived CYP metabolite, 2) activates BK_Ca channels and is linked to activation of myoendothelial gap junctions, and 3) is inhibited directly by NO in a reversible manner.

Epoxygenase-derived metabolites of AA have been shown to contribute greatly to BK- and AA-induced vasodilation in many vascular tissues including the rat kidney (5, 11, 16, 27), pig or dog coronary arteries (4, 22), and rat heart (12). In this study, we first obtained evidence for a role for epoxides in the dilator responses to AA and BK in preglomerular vessels using pharmacological inhibitors. The inhibition of AA- and BK-induced renal vasodilation by miconazole or PPOH, which are two mechanistically different epoxygenase inhibitors, is in agreement with that reported in other studies (11, 12, 16). Additional evidence for a role for epoxides was provided by experiments in which fixing epoxide levels with 11,12-EET, the most potent vasodilator epoxide of the afferent arteriole (16), diminished dilation produced by AA and BK but not that produced by hydralazine. This latter approach is considered more selective, as it avoids the problem of lack of specificity that is usually encountered with pharmacological CYP inhibitors. Epoxides have been demonstrated to be a putative EDHF in many vascular beds including kidney (5, 12, 14, 21). The hallmark of the vasorelaxation attributed to EDHF is that it is accompanied by membrane hyperpolarization (5, 7, 9, 23) with evidence that suggests that hyperpolarization generated in endothelial cells is capable of spreading electrotonically to the underlying smooth muscle most likely via myoendothelial gap junctions (31). The blockade by the gap junction inhibitor AGRA of the relaxation elicited by AA or BK supports a role for a functional gap junction in epoxide-mediated vascular relaxation in the renal microvessel. The attenuation by the BK_Ca channel inhibitor apamin of the dilation by AA or BK is in agreement with studies that demonstrate a role for K⁺ channels in epoxide-mediated vascular relaxation in kidney (30, 39). The attenuation by apamin of hydralazine-induced vasodilation is also consistent with the role of BK_Ca channels in its response (2). The blockade by AGRA of the dilation by hydralazine is not surprising considering that gap junctions are required for the spread of K⁺ currents between cells. Definitive evidence for a role for epoxides in the dilation elicited by AA or BK in renal microvessels was provided by the experiment in which we demonstrated that vessels incubated with AA or BK produce significant increases in epoxide production (Fig. 7). Because a role for epoxides is demonstrated and their effects are characterized in the renal dilator effects of AA and BK, the stage is set for evaluating NO-epoxygenase interactions in the kidney and the functional implications for regulation of renal vascular tone.

NO inhibits the activity of microsomal and purified CYP enzymes including CYP2C gene family members (34) that are responsible for generation of epoxides (18). Our laboratory and others have shown that NO donors inhibit renal microsomal epoxygenase activity (1, 28) and downregulate the expression of CYP2C isoforms (6, 34). In this study, inhibition by NO of CYP-dependent epoxygenase enzymes is demonstrated by attenuation by spermine NONOate or SNP of epoxide-mediated dilator effects of AA or BK in the renal microvessel. This is supported by the attenuation of basal and AA-stimulated production of epoxides by spermine NONOate in the renal microvessel. These data are consistent with the demonstrations that NO diminishes, whereas NO inhibition enhances, the expression of CYP2C (3, 9) and that NO inhibits BK-mediated production of EDHF in rabbit carotid and porcine conduit coronary arteries (3, 9, 22) and in the canine coronary vasculature (22, 23). Taken together, these observations support the notion that there are large increases in epoxide-hyperpolarizing factor-mediated responses in tissues in which NO production is inhibited (15); this suggests that NO exerts a tonic inhibitory effect on epoxygenase enzyme. However, this generalization must be qualified, because the available information supports the idea that this phenomenon may only apply to agonists such as BK and, perhaps, acetylcholine; for these agents, dilator effects involve NO and epoxides (11, 15, 16, 39, 40) but not AA, which has a predominantly epoxide-mediated dilator effect (23, 27).

Because NO activates cGMP in many cell systems, we evaluated the possibility that endothelial cGMP could regulate CYP enzymes in the renal microvessels. This does not appear to be the case: responses to AA or BK were unaffected by 8-bromo-cGMP, which is a cell-permeable analog of cGMP. This conclusion is similar to our earlier observation of renal microsomes in which SNP but not 8-bromo-cGMP inhibited NADPH-dependent conversion of [¹⁴C]AA to epoxygenase products (28).

NO inhibition of CYP enzymes was demonstrated to be initially reversible and due to the formation of nitrosyl complexes through binding to the heme moiety at the catalytic unit of CYP (41). However, an apparently irreversible inhibition that is due to nitration of tyrosine residues or oxidation of CYP protein thiols (36) has also been demonstrated. Our data do not support an irreversible inhibition by NO of renal epoxygenase activity inasmuch as the inhibition of AA-induced vasodilation was fully restored following addition of fresh Krebs buffer to the tissue that was previously exposed to NO donors.

Some studies have shown that NO possesses the capacity for hyperpolarizing vascular smooth muscle (24, 37) and that in rat renal arterioles, activation of BK_Ca channels contributes to 50% of the dilator response to NO (35). This being so, NO can therefore modulate responses to agonists through its effects on K⁺ channels. In this study, the lack of effect by NO of the dilation elicited by 11,12-epoxide, which is the most potent vasodilator epoxygenase arachidonate product of the afferent arterioles (16), the BK_Ca channel activator NS-1619 (8), or the K_ATP channel activator cromakalim, suggests that in the kidney, NO selectively inhibits epoxygenase production but not the actions of epoxides at K⁺ channels. This is at variance with the observation of the coronary microcirculation in which NO was demonstrated to inhibit the release as well as the actions of EDHF (3). Thus it appears that not only is NO-epoxygenase interaction agonist specific; the locus of inhibition also appears to be tissue specific. This notion is supported by the recent study of Schildmeyer and Bryan (33) in which NO did not show an effect on EDHF responses in rat cerebral vasculature.

In conclusion, we provide evidence that in rat preglomerular vessels, AA- or BK-induced vasodilation has a major epoxide component that is greatly diminished by NO but not 8-bromo-cGMP. The inhibition by NO of AA and BK dilation but the lack of effect of NO donors on dilation elicited by 11,12-EET and activators of K⁺ channels demonstrates a direct and specific inhibition by NO of the epoxygenase enzyme in the kidney. Thus as we demonstrated for 20-HETE in our previous studies (26), NO also probably provides a tonic inhibition of epoxygenase enzyme production, and in the absence of the tonic inhibition, the epoxygenase pathway may assume a predominant regulatory pathway in maintaining renal vascular tone.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-59884 and HL-03674. A. O. Oyekan is an Established Investigator of the American Heart Association.

	ACKNOWLEDGMENTS

 
The GC/MS facility of the Department of Pharmacology at New York Medical College was used for quantitative determination of epoxides.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. O. Oyekan, Center for Cardiovascular Diseases, College of Pharmacy and Health Sciences, Texas Southern Univ., 3100 Cleburne St., Houston, TX 77004 (E-mail: OYEKAN_AO{at}TSU.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 DISCLOSURES REFERENCES

 

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