Inhibitors of 20-Hydroxyeicosatetraenoic Acid Reduce Renal Vasoconstrictor Responsiveness

20-HETE is a cytochrome P450 (P450)-derived metabolite of arachidonic acid that is produced by vascular smooth muscle (Imig et al., 1996) where its constrictor activity is thought to relate to inhibition of K⁺ channels and stimulation of protein kinase C and L-type Ca²⁺ channels (Zou et al., 1996; Roman, 2002). Several studies have implicated 20-HETE as an integral component of vascular mechanisms such as the myogenic response and autoregulation of renal and cerebral blood flow (Kauser et al., 1991; Imig et al., 1994; Zou et al., 1994a,b). In addition, 20-HETE has been invoked as a second messenger contributing to the responses of some vasoactive hormones, including angiotensin II (Oyekan et al., 1997; Croft et al., 2000) and endothelin (Oyekan et al., 1997), both of which increased 20-HETE formation.

On the other hand, inhibition of 20-HETE synthesis has been proposed as a mechanism contributing to the vasodilator effects of NO donors (Alonso-Galicia et al., 1997, 1998). Thus, NO reduces 20-HETE formation by inhibiting ω-hydroxylase activity (Oyekan et al., 1999), and it was suggested that NO may serve as a tonic inhibitor of ω-hydroxylase. In support of this contention, inhibition of NO synthesis increased 20-HETE release (Oyekan et al., 1999). Accordingly, NO derived from NO donors would be expected to reduce the formation of 20-HETE, which, in turn, would increase the open probability of K⁺ channels and promote vascular relaxation. In contrast, removal of this inhibitory effect of NO by NO synthase inhibitors would increase the formation of 20-HETE, which could contribute to the vascular effects of these agents. Thus, inhibition of NOS enhances vascular resistance and increases responses to vasoconstrictor agents (Reid et al., 1991; Oyekan and McGiff, 1998).

Our primary objective was to address the role of 20-HETE in the renal vascular effects of removing NO in terms of increased vasoconstrictor responses and renal vascular resistance, to test the hypothesis that increased synthesis of 20-HETE contributes to the enhanced vasoconstrictor responses observed when NO synthesis is inhibited. To this end, we evaluated the effects of a highly specific ω-hydroxylase inhibitor, DDMS (Nguyen et al., 1999), on vasoconstrictor responses to U46619 in the absence and presence of nitroarginine (l-NA) to inhibit NO synthesis. These studies were extended to address the effects of inhibitors of K⁺ channels, the presumed target of 20-HETE, on renal vasoconstrictor responses. The results show that elevation of vascular tone by inhibition of NOS or addition of phenylephrine increased vasoconstrictor responses to U46619, effects prevented by inhibition of ω-hydroxylase. Similarly, inhibitors of large conductance Ca²⁺-activated K⁺ channels enhanced vasoconstrictor responses to U46619, supporting the suggestion that inhibition of K⁺ channels by increased 20-HETE formation could contribute to the enhanced vasoconstrictor responsiveness observed in the presence of NOS inhibitors. However, inhibition of 20-HETE reduced the enhanced responses to U46619 that were observed after K⁺ channel inhibition. Neither l-NA nor phenylephrine increased the release of 20-HETE from the kidney. Moreover, the inhibitor of ω-hydroxylase also reduced vasoconstrictor responses under basal conditions, suggesting that 20-HETE may contribute to vasoconstrictor responses.

Materials and Methods

Isolated Perfused Kidney

Male Wistar rats, 10 to 12 weeks of age, were used in these studies in accordance with National Institutes of Health guidelines. The isolated kidney was prepared as described previously (Fulton et al., 1992). Briefly, rats were anesthetized with pentobarbital (60 mg/kg i.p.) and the right kidney exposed by a midline laparotomy. The renal artery was cannulated via the mesenteric artery to avoid interruption of blood flow and the kidney perfused with oxygenated Krebs' buffer at 37°C at constant flow (10–12 ml/min) to obtain a basal perfusion pressure (PP) between 60 and 90 mm Hg. In some experiments where the perfusate was collected for the determination of 20-HETE release, the kidney was removed from the animal.

Experimental Protocols

Protocol 1. Once a stable PP was obtained, vasoconstrictor responses to U46619 (10–300 ng) were determined. After a 10- to 15-min interval, a second dose-response was compared to ascertain reproducibility and whether two dose-responses could be tested in the same preparation before and after any interventions. Because vasoconstrictor responses to U46619 were reproducible, i.e., consecutive dose-responses did not differ (Fig. 1), dose responses to U46619 were determined before and after any interventions.

Protocol 2. Responses to U46619 were determined in untreated kidneys and a second dose response was obtained after inhibition of NOS with l-NA (100 μM) with and without DDMS (10 μM) to inhibit ω-hydroxylase. Because the responses to U46619 did not differ during the first dose response (control) between the two groups (ANOVA), the data for the first dose-response were pooled. In some cases, 1-min perfusate collections were made immediately before and after administration of 30, 100, and 300 ng of U46619 during the control or pretreatment period and after treatment with either l-NA or l-NA plus DDMS for determination of 20-HETE release which was measured by GC/MS.

Protocol 3. Because l-NA increased PP, additional experiments were conducted to address vasoconstrictor responses to U46619 in the presence and absence of DDMS when PP was elevated with phenylephrine (7.5 × 10^–7 M) to the same level as that achieved with inhibition of NOS using the experimental design described under protocol 2. Responses to U46619 during the first dose response (control) were not different between the two groups (ANOVA), and the data were pooled.

Protocol 4. The effects of DDMS on vasoconstrictor responses to U46619 were also assessed at basal PP where NOS remained intact. Thus, dose responses to U46619 were obtained before and 10 to 15 min after the addition of DDMS (10 μM) to the renal perfusate.

Protocol 5. To support the results obtained with DDMS, we also used an antagonist of 20-HETE in a separate series of experiments. Thus, 20-HEDE (1 μM) exhibits antagonistic activity against 20-HETE at the concentration used here but also inhibits ω-hydroxylase at higher concentrations (Alonso-Galicia et al., 1999; Roman, 2002). As with DDMS, vasoconstrictor responses to U46619 were determined before and 10 to 15 min after the addition of 20-HEDE to the renal perfusate.

Protocol 6. In other experiments, responses to U46619 were compared before and 10 to 15 min after inhibition of K⁺ channels with TEA (1 mM), charybdotoxin (20 nM) and apamin (100 nM). At a concentration of 1 mM, TEA, a relatively nonselective inhibitor of K⁺ channels, is considered to be more selective for large conductance Ca²⁺-activated K⁺ channels, whereas charybdotoxin affects both large and intermediate conductance Ca²⁺-activated K⁺ channels and apamin inhibits small conductance Ca²⁺-activated K⁺ channels (Brayden and Nelson, 1992).

Protocol 7. Additional experiments designed to address the role of K⁺ channels in the regulation of vascular tone, were conducted where responses to bolus doses of TEA, charybdotoxin, and apamin were compared in untreated kidneys (basal PP) and those in which vascular tone was increased with U46619 (10 ng/ml), resulting in an elevation of PP to 95 to 110 mm Hg.

Protocol 8. In the final series of experiments, the effects of 20-HEDE (1 μM) on responses to U46619 were determined in the absence and presence of TEA (1 mM) to inhibit K⁺ channels. Thus, two groups were used, the first where dose responses to U46619 were determined before and after TEA and the second where dose responses to U46619 were obtained before and after combined treatment with TEA and 20-HEDE. Because responses to U46619 did not differ in the pretreatment period of either group, these data were pooled.

GC/MS Analysis of 20-HETE. In some cases, 1-min perfusate collections were made immediately before and after the administration of each dose of U46619 during the first (pretreatment) and second (treatment) dose responses. Deuterated 20-HETE (100 pg/ml) was added to 10 ml of each sample as an internal standard. The samples were acidified with acetic acid to pH 4 and the lipids extracted twice with 5 ml ethyl acetate, which was decanted and dried. The sample was reconstituted in 50 μl of methanol for reverse phase high-performance liquid chromatography purification of 20-HETE using an HP 1050 instrument with a Beckman ODS column (25 cm × 4.6 mm, 5 μm) and a linear gradient of 60 to 100% acetonitrile containing 0.025% acetic acid over 20 min at a flow rate of 1 ml/min. The peak corresponding to authentic 20-HETE was collected, the sample dried, and the 20-HETE derivatized to pentafluorobenzyl esters and trimethylsilyl ethers. Pentafluorobenzyl esters were prepared by the addition of 30 μl of 10% α-bromo-2,3,4,5,6-pentafluorotoluene (Aldrich Chemical Co., Milwaukee, WI) in acetonitrile and 30 μl of 10% N,N-diisopropylethylamine (Aldrich Chemical Co.) in acetonitrile. After 30 min at room temperature, the samples were dried and the trimethylsilyl ethers prepared by dissolving the samples in 60 μl of N,O-bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich) and 20 μl of pyridine and allowing the reaction to proceed for 30 min at room temperature. The samples were dried and dissolved in 50 μl of isooctane and 1-μl aliquots were analyzed using an HP 5890 GC/MS. The gas chromatograph column (DB-1; 10 m, 0.25 mm inner diameter, 0.25-μm film thickness; Agilent Technologies, Palo Alto, CA) was temperature programmed from 180–300°C at a rate of 25°C/min. Methane was used as a reagent gas at a flow resulting in a source pressure of 2 torr and the mass spectrometer (HP 5989A) was operated in the electron capture chemical ionization mode and monitoring ions at m/z of 391 and 393, representing the derivatives of the unlabeled and d2-labeled 20-HETE. The amount of 20-HETE in the samples was calculated by reference to a standard curve.

Analysis. In most of the experiments, each kidney served as its own control where responses to U46619 were determined before and after any interventions, and data were compared by ANOVA with a modified t-statistic (Bonferroni) for individual points. p < 0.05 was considered statistically significant.

Materials. U46619 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA), dissolved in ethanol, and diluted with water to 100 μg/ml; 100-μl aliquots were stored at –20°C. Apamin, TEA, and phenylephrine were obtained from Sigma-Aldrich and dissolved in water. Charybdotoxin was obtained from Peptides International Inc. (Louisville, KY) and dissolved in water. DDMS and 20-HEDE were prepared by Dr. J. R. Falck (University of Texas Southwestern Medical Center, Dallas, TX) and stored as stock solutions in ethanol at –20°C.

Results

Initial experiments were conducted to assess the reproducibility of consecutive dose-responses in the same kidney to determine whether comparisons could be made before and after any interventions. Figure 1 shows that U46619 (10, 30, 100, and 300 ng) produced comparable increases in renal PP upon repeated administration to the same kidney where the dose responses were separated by approximately 15 min. Consequently, subsequent experiments used two dose responses where the first served as a control for the second, which was obtained after one of several interventions.

Inhibition of NOS with l-NA increased PP in the kidney to 190 ± 7 mm Hg from 79 ± 3 mm Hg whereupon vasoconstrictor responses to U46619 were enhanced, the greatest effect being observed at the lower doses of U46619; the increases in PP in response to 10 and 30 ng were doubled in the presence of l-NA (Fig. 2). In the presence of DDMS to inhibit ω-hydroxylase, the l-NA-induced enhancement of vasoconstrictor responses to U46619 was prevented (Fig. 2). Thus, 30, 100, and 300 ng of U46619 raised PP by 14 ± 6, 31 ± 8, and 55 ± 2 mm Hg, respectively, values very similar to those obtained in kidneys not exposed l-NA. DDMS did not affect the increase in PP induced by l-NA, 71 ± 5 mm Hg in control versus 179 ± 5 mm Hg after l-NA plus DDMS, questioning the contribution of 20-HETE to the vasoconstriction resulting from inhibition of NO synthesis.

One interpretation of the results with DDMS is that 20-HETE contributes to the vasoconstrictor effects of U46619. However, measurements of 20-HETE released into the renal perfusate showed that U46619 did not increase the release of 20-HETE, which exhibited an initial time-dependent increase during the first dose response to U46619 (Fig. 3). The release of 20-HETE during the control dose-responses for l-NA alone and l-NA plus DDMS was remarkably similar (Fig. 3, top and bottom, before any treatment). Upon treatment with l-NA to increase PP, release of 20-HETE did not increase as expected (Fig. 3, top). Inhibition of ω-hydroxylase with DDMS was associated with a decrement in 20-HETE release relative to that from kidneys treated with l-NA alone (Fig. 3, bottom versus top).

To examine whether the effect of l-NA in enhancing responses to U46619 was related to the increase in PP rather than removal of an NO-mediated inhibitory influence on 20-HETE generation, we also tested responses to U46619 after elevation of PP from 79 ± 5 to 185 ± 5 mm Hg with phenylephrine. Under conditions of elevated PP, vasoconstrictor responses to U46619 were enhanced (Fig. 4) to a similar degree as with l-NA. Thus, responses to 10, 30, and 100 ng of U46619 were increased approximately 2-fold. In the presence of DDMS where PP was elevated from 71 ± 3 to 157 ± 6 mm Hg, the increases in the responses were abolished and the responses to 100 and 300 ng U46619 were reduced compared with the corresponding control values.

Because DDMS reduced the enhanced vasoconstrictor responses to U46619 after elevation of PP induced by either l-NA or phenylephrine and suggested a generalized effect, we also determined the effects of DDMS on vasoconstrictor responses to U46619 under conditions of basal PP. Similar to the effects observed when PP was elevated, DDMS reduced the vasoconstrictor effects of U46619 (Fig. 5), shifting the dose-response curve to the right. DDMS did not affect basal PP.

In an attempt to discern effects of DDMS on vasoconstrictor responses that were independent of 20-HETE, we also tested the effects of a 20-HETE antagonist, 20-HEDE (1 μM), on vasoconstrictor responses to U46619. Under conditions of basal PP, which was unaffected by 20-HEDE, inhibition of 20-HETE with 20-HEDE also reduced the vasoconstrictor effects of U46619 (Fig. 6), shifting the dose response to the right.

Because one of the targets for 20-HETE is considered to be the large conductance Ca²⁺-activated K⁺ channel, we reasoned that known inhibitors of this type of K⁺ channel should produce effects opposite to those of inhibitors of 20-HETE and enhance vasoconstrictor responses. In a separate series of experiments, we addressed the effects of K⁺ channel inhibitors on renal vasoconstrictor responses to U46619 under conditions of basal PP. Both TEA and charybdotoxin (Fig. 7) markedly enhanced vasoconstrictor responses to U46619, whereas apamin was without effect (data not shown), suggesting that activation of large conductance, but not small conductance, Ca²⁺-activated K⁺ channels moderate vasoconstrictor responses. Neither charybdotoxin nor TEA altered renal PP.

In further experiments to assess the role of K⁺ channels in the regulation of vascular tone, we tested the vasoconstrictor effects of K⁺ channel inhibitors. At basal PP (60–90 mm Hg), bolus doses of TEA (1–5 mg) or charybdotoxin (0.1–3 μg) produced little effect on PP (Fig. 8). However, when vascular tone was elevated with U46619 to raise PP to 95 to 110 mm Hg, both TEA and charybdotoxin elicited dose-dependent increases in PP (Fig. 8), indicating that K⁺ channel activity was increased in the presence of U46619.

To further investigate the contribution of K⁺ channels to the effects of the 20-HETE inhibitors on vasoconstrictor responses, we determined the effect of 20-HEDE on the enhanced vasoconstrictor responses to U46619 resulting from inhibition of K⁺ channels with TEA. As before, inhibition of K⁺ channels with TEA, which did not affect PP, greatly enhanced the vasoconstrictor responses to U46619 (Fig. 9). In the presence of 20-HEDE and TEA, vasoconstrictor responses to U46619 were not different from those obtained under control conditions, i.e., first dose response. These results suggest that inhibitors of 20-HETE do not moderate vasoconstrictor responses by influencing the activity of K⁺ channels.

Discussion

20-HETE is a P450-dependent metabolite of arachidonic acid that is produced by the preglomerular vasculature (Marji et al., 2002). This eicosanoid is a vasoconstrictor agent that has been implicated in mechanisms controlling glomerular filtration rate and renal blood flow (Kauser et al., 1991; Imig et al., 1994; Zou et al., 1994a,b) and as a component of vasoconstrictor mechanisms stimulated by vasoactive hormones such as AII and endothelin (Oyekan et al., 1997; Croft et al., 2000), which stimulate the vascular production of 20-HETE (Croft et al., 2000). Thus, 20-HETE inhibits the activity of maxi K⁺ channels, leading to depolarization of the membrane, influx of Ca²⁺ and contraction of vascular smooth muscle (Roman, 2002). In addition to its role in vasoconstrictor mechanisms, 20-HETE has also been implicated in vasodilation produced by NO donors (Alonso-Galicia et al., 1997, 1998). Thus, NO inhibits P450-dependent enzymes and reduces the formation of 20-HETE (Oyekan et al., 1999). It has been proposed that removal of a tonic inhibitor of K⁺ channels would increase the open probability of these channels leading to hyperpolarization and promotion of smooth muscle relaxation. In this scheme, inhibition of NO synthesis would increase the formation of 20-HETE to promote smooth muscle contraction. An increase in 20-HETE formation after inhibition of NO synthesis with l-NAME has been implicated in the increases in vascular resistance and blood pressure induced by this agent (Oyekan and McGiff, 1998). Because inhibitors of NO synthesis would also be expected to increase responsiveness to vasoconstrictor agents, the primary aim of the present study was to test the hypothesis that increased synthesis of 20-HETE contributes to the enhanced vasoconstrictor responses observed when NO synthesis is compromised.

First, we showed that inhibition of NO formation with l-NA increased vasoconstrictor responsiveness to U46619 in the isolated perfused kidney of the rat and that this effect was prevented by an inhibitor of 20-HETE synthesis, DDMS, which is specific for ω-hydroxylase (Nguyen et al., 1999). The results with DDMS implicate 20-HETE in the enhanced vasoconstrictor effect of U46619 when NO formation is inhibited. However, we were unable to show increased efflux of 20-HETE into the renal perfusate subsequent to inhibition of NOS, arguing against a role of increased synthesis of 20-HETE. Moreover, the increase in vascular resistance resulting from NOS inhibition and reflected by an increase in PP does not seem to involve increased formation of 20-HETE because DDMS did not modify the elevation in PP. If the vasoconstrictor effect of l-NA reflects, in part, an increase in de novo synthesis of 20-HETE, then inhibition of synthesis might be expected to attenuate the vasoconstrictor effect of l-NA. However, an effect of inhibition of 20-HETE may be more apparent if the inhibitor was added before the elevation of PP with l-NA.

We addressed whether an elevation in PP, per se, would increase vasoconstrictor responsiveness by using phenylephrine to increase PP to similar levels as those attained with l-NA. Increasing PP with phenylephrine increased vasoconstrictor responses to U46619, an effect also prevented by the presence of DDMS and, thereby, implicating 20-HETE. However, 20-HETE does not seem to be a mediator of the response to U46619 because U46619 did not increase the renal release 20-HETE, either under control conditions or after treatment with l-NA. In light of the observations that DDMS reduced 20-HETE efflux by approximately 50% in l-NA-treated kidneys, coincident with restoration of vasoconstrictor responsiveness to control levels, we suggest that 20-HETE may contribute to receptor-mediated vasoconstrictor mechanisms. This suggestion receives support from the observations that, under basal conditions, DDMS also led to a reduction in the vasoconstrictor responses to U46619. Further support derives from the studies in interlobar arteries by Kaide et al. (2000), who demonstrated that inhibitors ω-hydroxylase shift the dose response for phenylephrine to the right, an effect associated with reduced 20-HETE formation and reversed by the addition of exogenous 20-HETE.

An alternative explanation for our results is that DDMS impaired vasoconstrictor responses by actions unrelated to inhibition of ω-hydroxylase and 20-HETE formation. However, this possibility was countered by results showing that 20-HEDE, an antagonist of the vasoconstrictor actions of 20-HETE in renal and cerebral blood vessels (Alonso-Galicia et al., 1999; Gebremedhin et al., 2000), also shifted the dose response for U46619 to the right, producing a similar level of inhibition of vasoconstrictor responses to U46619 as DDMS. These results lend additional support for a role of 20-HETE as a component of vasoconstrictor mechanisms, possibly resulting from its ability to reduce the activity of K⁺ channels.

Activation of Ca²⁺-activated K⁺ channels would be expected in response to vasoconstrictor agents or stimuli that increase intracellular Ca²⁺ (Brayden and Nelson, 1992). The increase in K⁺ channel activity in vascular smooth muscle would serve to limit the depolarization and reduce the activity of voltage-sensitive Ca²⁺ channels and the influx of Ca²⁺, and thereby, the contractile response. Thus, any agent that reduced the open probability of these channels might be expected to enhance vasoconstrictor responses by reducing the operation of a mechanism that serves to moderate vasoconstriction. Because 20-HETE has been shown to target large conductance Ca²⁺-activated K⁺ channels, we tested the effects of inhibitors of Ca²⁺-activated K⁺ channels that should mimic the effects of 20-HETE and enhance constrictor responses. This proposition was confirmed because both charybdotoxin and TEA, which was used at a concentration considered selective for large conductance Ca²⁺-activated K⁺ channels, markedly enhanced the vasoconstrictor effect of U46619 and lowered the threshold. The role of Ca²⁺-activated K⁺ channels in moderating responses to vasoconstrictor agents was confirmed by showing that bolus administration of TEA or charybdotoxin elicited dose-dependent vasoconstrictor responses when the renal vasculature was slightly constricted with U46619 but had little vasoconstrictor activity in kidneys perfused at basal PP. In contrast, there was no evidence of a role for small conductance Ca²⁺-activated K⁺ channels in moderating vasoconstrictor responses to U46619 because there was no effect of apamin. This is consistent with the lack of activity of 20-HETE at small conductance K⁺ channels.

In our interpretation of these data, we have made the assumption that the effects of the K⁺ channel inhibitors reflect actions on vascular smooth muscle. However, we cannot exclude the possibility that TEA and charybdotoxin and, possibly, 20-HETE produce some of their effects by interacting with endothelial K⁺ channels. Activation of endothelial K⁺ channels leads to increased influx of Ca²⁺ that should result in activation of pathways that lead to the release of vasodilator mediators such as NO, prostaglandins, and epoxyeicosatrienoic acids. If vasoconstrictor stimuli lead to activation of endothelial K⁺ channels, which, in turn, result in the formation of vasodilator substances to moderate the vasoconstrictor response, then inhibition of these channels could be expected to also enhance vasoconstrictor responses.

Because 20-HETE reduces the activity of K⁺ channels and established inhibitors of K⁺ channels enhance vasoconstrictor responses, it is possible that increased activity of K⁺ channels, resulting from removal of an inhibitor, contributes to the effects of inhibitors of 20-HETE. If this were the case, then inhibitors of 20-HETE would be expected to have little effect on the enhanced vasoconstrictor responses induced by K⁺ channel blockade. However, we found that 20-HEDE reduced vasoconstrictor responses to U46619 in the presence of TEA, indicating that increased K⁺ channel activity does not contribute to the actions of inhibitors of 20-HETE in reducing vasoconstrictor responses to U46619. An alternative mechanism may involve effects on Ca²⁺-dependent processes because the vasoconstrictor effect of 20-HETE in porcine coronary arteries depends, in part, on sensitization of the contractile apparatus to Ca²⁺ (Randriamboavonjy et al., 2003).

In summary, our data indicate a contribution of 20-HETE to vasoconstrictor responses to U46619 as the enhanced responses observed when PP was elevated by either l-NA or phenylephrine were prevented by an agent that reduced 20-HETE release or antagonized its actions. Although inhibitors of K⁺ channels enhance vasoconstrictor responses, any contribution of 20-HETE to vasoconstrictor responses does not seem to result from its ability to inhibit K⁺ channels.