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Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca²⁺ changes and dilation in isolated resistance arteries

Physiologisches Institut, Ludwig Maximilians Universität, München, Germany; and
^* Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe Universität, Frankfurt, Germany

¹Correspondence: Physiologisches Institut, Ludwig Maximilians Universität, Schillerstrasse 44, D-80336 München. E-mail: bolz{at}lrz.uni-muenchen.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Using a novel vessel culture technique in combination with antisense oligonucleotide transfection, we tested whether the endothelium-derived hyperpolarizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arteries from hamster gracilis muscle (n=19) were perfused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in endothelial cells. Thereafter, NO- and prostaglandin-independent, EDHF-mediated vascular responses associated with hyperpolarization [i.e., decrease in smooth muscle calcium (Fura 2) and vasodilation] were studied after the application of acetylcholine (ACh). These EDHF-mediated responses were markedly attenuated (by 70%) by As- but not by S- or Scr-oligonucleotide treatment. However, the responses to norepinephrine (0.3 µmol/l), the NO donor sodium nitroprusside (1 µmol/l), and the K_Ca channel activator NS1619 (100 µmol/l) were unaltered. As treatment, which specifically targeted the endothelial layer (as assessed by confocal microscopy), had no inhibitory effect on increases in endothelial calcium to ACh. It is concluded that a CYP2C8/9-related isoform functions as an EDHF synthase in hamster resistance arteries and that a product of this enzyme is an EDHF, or at least an integral part of the signaling cascade leading to EDHF-mediated responses.—Bolz, S.-S., Fisslthaler, B., Pieperhoff, S., de Wit, C., Fleming, I., Busse, R., Pohl, U. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca²⁺ changes and dilation in isolated resistance arteries.

Key Words: organ culture • cytochrome P450 metabolites • hyperpolarization • endothelium • acetylcholine

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE ENDOTHELIUM-DEPENDENT DILATION in response to acetylcholine (ACh) is partially due to a hyperpolarization of vascular smooth muscle cells that is not mediated by nitric oxide (NO) or prostacyclin (PGI₂). This NO/PGI₂-independent vascular response, which is especially prominent in resistance arteries, is generally attributed to the release of a separate ‘endothelium-derived hyperpolarizing factor’ (EDHF) (1) . From the experimental data published to date, it is evident that several EDHFs exist in different species (2 , 3) . However, the majority of reports characterizing EDHF in the microcirculation support the concept that this EDHF is a cytochrome P450 (CYP)-related product. This hypothesis was based on the observation that CYP inhibitors such as 17-ODYA (4) and a number of anesthetics (5 , 6) markedly attenuate EDHF-mediated dilations. However, these conclusions are limited by the fact that the above-mentioned CYP inhibitors cannot discriminate between the different CYP isoforms, and that imidazole-containing CYP inhibitors can directly interfere with K⁺ channels (7) , which are the main targets of EDHF in vascular smooth muscle cells (8) .

In previous studies, several products of the cytochrome P450 pathway (hydroxyeicosatrienoic, di-hydroxyeicosatrienoic, and epoxyeicosatrienoic acids) have been shown to hyperpolarize vascular smooth muscle cells by activation of different K⁺ channels (9 10 11 12) . On the other hand, the CYP-epoxygenase product of arachidonic acid 11,12-epoxyeicosatrienoic acid (11,12-EET) activates calcium-dependent K⁺ channels (K_Ca channels) (13) and elicits smooth muscle relaxation. 11,12-EET is synthesized by the CYP isoforms 2C8, 2C9, and 1A2 (14) , and 2J (15) . Of these isoforms, CYP2C8 is a candidate ‘EDHF synthase’ as it is expressed in human endothelial cells (16) .

The aim of the present study was to clarify the involvement of CYP2C8/9 in EDHF-mediated responses in small skeletal muscle resistance arteries using a novel nonpharmacological approach based on antisense oligonucleotide transfection. We determined whether the reduction of endothelial CYP 2C as a consequence of transfection with an antisense oligonucleotides against the coding region of CYP 2C8/9 would selectively affect EDHF-mediated effects in response to acetylcholine in resistance arteries.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Preparation of small resistance arteries
Female golden Syrian hamsters (149±2 g body weight) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). Segments of small resistance arteries (resting outer diameter 234±8 µm) were excised under sterile conditions from the gracilis muscle, cannulated with glass micropipettes and transferred to an organ bath.

Determination of oligonucleotide sequence
The sequence of the oligonucleotide was derived from the cDNA sequences of the cytochrome P450 epoxygenases 2C8 and 2C9 (5'GAGGAGTGGGGCCAGGAGGGAG 3'). It is located 50 bp downstream of the translation initiation site within the coding region. Comparison of the oligonucleotide sequence with the database detected only homology to cytochrome P450 sequences from the 2C family, including the 2C of the Syrian hamster. To detect the localization within the vessel, the 5' ends of the oligonucleotides were conjugated to fluorescein-isothiocyanate (FITC). Sense and scrambled oligonucleotides were synthesized for use as controls.

RNA isolation and reverse transcriptase-polymerase chain reaction
Arteries and heart tissue from Syrian hamster were frozen in liquid nitrogen and RNA was extracted by guanidine isothiocyanate and phenol (Trizol, Life Technologies, Inc., Eggenstein, Germany). Random hexanucleotide primers were used for reverse transcription of equal amounts of RNA, and the oligonucleotides used for polymerase chain reaction (PCR) were derived against a human CYP2C8 sequence (genebank accession no. Y00498; upstream primer: GATAGATGTTAAGGACATCT, downstream primer: CTCCCTAATGTAACTTCGTG) that exhibited a high homology to the Syrian hamster 2C25 sequence (genebank accession no. D11435). After PCR, the fragments were separated on a 1.5% TAE agarose gel and visualized by staining with ethidium bromide. To verify the DNA fragment, PCR products were transferred to nylon membranes and hybridized with ³²P-labeled DNA fragments encoding 2C8.

Organ culture and transfection
The segments were per- and superfused at a rate of 1 ml/h with culture medium supplemented with 15% heat-inactivated cool calf serum, 20,000 u/l penicillin, and 20 mg/l streptomycin. The transmural pressure was maintained at 45 mmHg. A 3 ml perfusion medium containing 2.5 µg/ml FITC-labeled antisense, sense, or scrambled oligonucleotides and 20 µl/ml transfection reagent (Superfect; Qiagen, Chatsworth, Calif.) was applied to the segments and left in contact for 3 h. Thereafter, the segments were kept in organ culture for 24–32 h. The segments were then transferred to the stage of a laser scanning microscope (Zeiss, LSM 410) to localize FITC fluorescence within the vascular wall. The segments were then incubated with the Ca²⁺-sensitive dye fura 2 [2 µmol/l fura 2-acetoxymethylester (AM) and 0.5% bovine serum albumin (BSA) in MOPS-buffered salt solution]. To facilitate selective measurements of smooth muscle Ca²⁺_i, fura 2-AM was added to the organ bath; selective loading of the endothelium (EC) could be achieved by perfusion of the segments with fura 2-AM. Ca²⁺_i in VSMC or EC was measured as described previously in more detail (17) . Video microscopy was performed at wavelengths > 610 nm, which did not interfere with the fluorescence signal emitted by fura 2.

Experimental protocol
All segments were treated with indomethacin (30 µmol/l) and constricted with norepinephrine (NE, 0.3 µmol/l, 2 min) prior to the addition of vasodilators. ACh was applied to the organ bath, and subsequent changes of Ca²⁺ and outer diameter were recorded for 2 min. To separate NO- and EDHF-mediated effects of ACh, vascular responses were compared before and after treatment with the inhibitor of NO synthase (NOS), Nnitro-L-arginine (L-NA, 30 µmol/l). To establish that the effects of ACh persisting after L-NA treatment were mediated by EDHF, charybdotoxin (ChTX, 1 µmol/l), a specific inhibitor of calcium-activated potassium channels (K_Ca channels), was applied. Changes in Ca²⁺_i and diameter mediated by EDHF in response to ACh were compared in segments treated with As-, S-, or Scr-oligonucleotides. To exclude unspecific effects of the transfection procedures, endothelium-independent vasodilatory effects of sodium nitroprusside (SNP) were also studied. Moreover, the responses of cultured vessels were compared with those of freshly isolated resistance arteries to control for unspecific effects of the organ culture protocol.

Drugs and solutions
The organ bath solution used for functional studies contained (mol/l) 145 NaCl, 4.7 KCl, 1.5 CaCl₂, 1.17 MgSO₄, 1.2 NaH₂PO₄, 2.0 pyruvate, 0.02 EGTA, 3.0 MOPS, and 5.0 glucose. NE, ACh, L-NA, ChTX, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), and SNP were from Sigma Chemical (Deisenhofen, Germany), MnCl₂ from Merck (Darmstadt, Germany), and fura 2-AM from Molecular Probes (Eugene, Oreg.). Drugs were dissolved in MOPS buffer, Na-acetate (SNP), or DMSO (fura 2-AM). Stock solutions were further diluted to five times that of the final concentration. For the application, one-fifth of the bath volume was exchanged to achieve the final bath concentration to which we refer throughout in the text.

Statistical analysis
Dilations are expressed as % dilator capacity = ((dia_vasodilator-dia_NE)/(dia_max-dia_NE)) x 100, where dia_vasodilator represents the steady-state outer diameter 2 min after stimulation with the respective vasodilator, where dia_NE is the steady-state outer diameter on stimulation with NE, and dia_max is the maximal diameter of the resistance arteries at 45 mmHg. Due to potential errors associated with measurements in an intact vessel (18 ), Ca²⁺_i in vascular smooth muscle cells can only be estimated. We therefore used ratio changes to describe % changes in Ca²⁺_i. According to calibration curves obtained in a cell-free system, the range of ratios observed here (0.4–36) fitted well into the linear range of the calibration curve (42.2 to 1520 nmol/l), which is a prerequisite for calculating % changes. Changes in Ca²⁺_i are expressed as Ca²⁺_i, from ((R_NE-R_vasodilator)/(R_NE-R_basal)) x 100. The indices for the different R values correspond to those used for the diameter measurements except for R_basal, which describes the calcium level under unstimulated conditions.

All results are presented as means ± SE of n experiments, n representing the number of vessels used per experiment. Steady-state values of different experimental groups were compared using Student’s t test. Differences were considered significant at error probabilities less than 0.05 (P<0.05).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
EDHF-mediated responses elicited by acetylcholine in resistance arteries
Acetylcholine induced dose-dependent dilations of resistance arteries preconstricted using NE to 35 ± 1% of their maximal diameter (n=8). These dilations were preceded by a marked decrease in smooth muscle Ca²⁺_i. Treatment with L-NA reduced the dilation elicited by lower ACh doses without affecting the Ca²⁺ decrease in vascular smooth muscle cells (Fig. 1 ). The dilation induced by the highest concentration of ACh was, however, completely insensitive to L-NA. Again, decreases in Ca²⁺_i as well as ACh-induced hyperpolarizations (data not shown) remained unaffected by L-NA. All of the L-NA/indomethacin-insensitive responses were abolished by the inclusion of ChTX, a specific inhibitor of K_Ca channels (Fig. 1) , or by the removal of the endothelium (n=5, data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effects of increasing concentrations of acetylcholine (ACh, 0.01–1 µmol/l) on smooth muscle calcium (Ca2+i, top) and vascular diameter ( diameter, bottom) in indomethacin-treated resistance arteries (n=8) that were preconstricted with norepinephrine (NE, 0.3 µmol/l). The same arteries were studied before and after treatment with the NOS inhibitor L-NA and subsequently the KCa channel inhibitor charybdotoxin (ChTX, 1 µmol/l). Ca2+i (%) refers to the reversal of NE-induced increase in smooth muscle Ca2+. diameter refers to % of dilator capacity. Values are means ± SE. *P < 0.05, significantly different from dilations and decreases in Ca2+i in untreated vessels.

NE-induced contractions as well as ACh-induced EDHF- and NO-mediated dilations were not significantly different in freshly isolated segments and segments kept under organ culture conditions for up to 48 h (19) .

Expression of CYP2C in aorta and A. gracilis
Reverse transcribed RNA from hamster aorta and A. gracilis was analyzed for the expression of CYP2C-related genes. PCR primers derived from a human CYP2C8 sequence homologous to hamster 2C25–27 resulted in the amplification of a fragment with the expected size of 616 bp (Fig. 2A ). A strong cross-hybridization with a DNA fragment encoding CYP2C8 confirmed the identity of the amplification product (Fig. 2B ). Amplification of reverse transcribed RNA from hamster liver and heart known to express large amounts of CYP2C8 with the same oligonucleotides resulted in fragments of identical size (data not shown).

View larger version (113K): [in this window] [in a new window]   Figure 2. CYP2C-related genes are expressed in hamster resistance arteries and aorta. RT-PCR was used to amplify CYP2C-related mRNA (A) and GAPDH (C) from segments of the aorta (aorta) and the A. gracilis (A.g.). Hybridization with a DNA fragment encoding CYP2C8 confirmed the identity of the PCR amplification product (B). After size fractionation of the PCR fragments, the agarose gel was stained with ethidium bromide. n.c. = negative control.

Targeting of oligonucleotides to the endothelium
FITC-labeled oligonucleotides were used to assess the selectivity and efficiency of the transfection protocol. Untreated resistance arteries emitted no fluorescence when excited at 488 nm. In contrast, marked fluorescence signals could be detected in resistance arteries transfected with either FITC-labeled antisense, sense, or scrambled oligonucleotides. The FITC-related fluorescence was homogenously distributed and confined to the endothelial layer (Fig. 3A, B ). The labeled structures were spindle-shaped and orientated along the longitudinal axis of the vessel, consistent with the orientation of endothelial cells in a native segment. In a separate series, de-endothelialized vessels were exposed to FITC-labeled oligonucleotides. In these vessels, the labeled structures were perpendicularly oriented as is typical for vascular smooth muscle cells (data not shown).

View larger version (72K): [in this window] [in a new window]   Figure 3. Distribution of FITC-labeled antisense oligonucleotides (green fluorescence) within the vascular wall. Confocal slices were taken at the bottom (A) and midline (B) of the vessel. Fluorescence images (green) were projected on the video image of the vessel. The distribution of FITC-labeled oligonucleotides was similar in sense and scrambled oligonucleotide-treated vessels.

Effects of oligonucleotide treatment on EDHF-mediated responses
To ensure the specific effect of the antisense oligonucleotide treatment on EDHF-mediated responses, experiments were also performed with sense (n=6) or scrambled (n=6) oligonucleotides (Fig. 4 ). The constriction induced by NE was similar in sense, scrambled, and antisense-treated segments (41±2, 45±1, 43±1%, n=6–12) as was the increase in smooth muscle Ca²⁺_i (41±3, 39±4, 43±5%). ACh (1 µmol/l) reversed the NE-induced increase in smooth muscle Ca²⁺_i to a similar extent in untreated segments as well as in those treated with sense and scrambled oligonucleotides. Accordingly, the resulting dilations in sense (86±3%) dilator capacity and scrambled (77±6%) treated segments were not significantly different from those in untreated segments (87±7%).

View larger version (42K): [in this window] [in a new window]   Figure 4. Left: Effects of oligonucleotide treatment (antisense, sense, and scrambled) on acetylcholine (ACh 1 µmol/l), NS1619 (100 µmol/l), and sodium nitroprusside (SNP 1 µmol/l) -induced decreases in smooth muscle Ca2+i and dilations (n=6–7). Vessels were pretreated with indomethacin (30 µmol/l) and L-NA (30 µmol/l). Values are means ± SE. *P < 0.05, significantly different from control (sham). Right: Original recordings showing the effect of antisense (top) or sense (bottom) oligonucleotides on the acetylcholine (ACh, 1 µmol/l) -induced increase in endothelial Ca2+i in intact segments. Similar results were obtained in two additional experiments.

In contrast, treatment with antisense oligonucleotides significantly reduced the EDHF-mediated decreases in smooth muscle Ca²⁺_i and subsequent dilations whereas the responses to the specific K_Ca channel activator NS1619 (100 µmol/l; Fig. 4 , left) were not altered. SNP-induced dilations were also similar in antisense and sense oligonucleotide-treated segments (Fig. 4 , left). Antisense oligonucleotide treatment did not affect endothelial increases in Ca²⁺_i in response to ACh.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
With the aid of antisense oligonucleotides against the coding region of CYP2C8/9, we have demonstrated the involvement of this CYP isoform in the EDHF-mediated responses of a resistance artery. Although the concept that a CYP-dependent metabolite of arachidonic acid is an EDHF has been proposed before, the nonselective nature of the CYP inhibitors currently available in some cases masked the actions of EDHF as a consequence of their direct action on K_Ca channels. Moreover, these compounds cannot be used to differentiate between the various CYP activities (e.g., epoxygenase vs. hydroxylase) or provide detailed information on the nature of the CYP-related EDHF synthase. The method described here uses antisense transfection techniques and targets a CYP isoform that displays the characteristics of the CYP-related EDHF synthase in that it is an isoform expressed in endothelial cells, the expression of which can be enhanced by ß-naphthoflavone. This compound has previously been shown to increase the production of a CYP-related EDHF by cultured human endothelial cells and porcine coronary arteries (20) . CYP2C8 is the 2C isoform isolated and cloned from human endothelial cells (16) and corresponds, for example, to 2C11 in the rat (21) and 2C25–27 in the hamster (22) . All of these isoforms are expressed in endothelial cells and produce the stereoisomers of epoxy-eicosatrienoic acid (11,12-, 14,15-, and 5,6-EET) via epoxidation of arachidonic acid. 14,15- and 11,12-EETs have been shown to activate charybdotoxin-sensitive K_Ca channels in vascular smooth muscle cells, which in turn mediate the effects of EDHF in resistance arteries of the hamster (8) . That EDHF mediates the NO/PGI₂-independent effects observed in the present study is suggested by our previous findings that both the Ca²⁺ decrease as well as the ACh-induced dilation and hyperpolarization are highly sensitive to charybdotoxin.

The inhibitory effect of the CYP2C8/9 antisense oligonucleotide was selective, as it attenuated EDHF-mediated dilations and decreases in smooth muscle Ca²⁺_i without affecting the responses to the K_Ca channel opener NS1619, endothelial Ca²⁺ signaling, or vascular reactivity to norepinephrine and SNP. Previous studies have suggested that CYP metabolites play a determining role in the regulation of the capacitative Ca²⁺ entry in endothelial cells (23 , 24) . As an increase in endothelial Ca²⁺ is thought to precede the activation of PLA₂, liberation of arachidonic acid, and activation of the EDHF synthase, it was essential to exclude an effect of the antisense oligonucleotides on endothelial Ca²⁺ signaling. Indeed, neither the sense nor the antisense oligonucleotides had a significant effect on the acetylcholine-induced increase in endothelial Ca²⁺. Thus, an attenuated Ca²⁺ response to acetylcholine cannot account for the effects of CYP2C8/9 antisense oligonucleotides on EDHF-mediated responses. The inhibition of EDHF-mediated dilation cannot be explained by a general impairment of smooth muscle or K_Ca channel function after oligonucleotide transfection. This statement is based on the fact that the NE-induced constriction and NS1619- and SNP-induced dilations were similar in all of the treatment groups, and is further supported by the finding that no FITC fluorescence was found in smooth muscle cells of vessels treated with FITC-labeled oligonucleotides. The selective targeting of endothelial cells under the experimental conditions used is not remarkable, since it is possible to selectively load endothelial cells with lipophilic compounds such as fluo-3 AM (25) and di-ANEPPS-AM (26 , 27) .

In summary, we have used a novel organ culture technique in combination with oligonucleotide transfection to test the hypothesis that CYP2C8/9 is an EDHF synthase. Although our approach does not distinguish between a direct hyperpolarizing action of a CYP 2C8/9 metabolite or its possible permissive role for the actions of a different EDHF, it demonstrates that a product of CYP2C8/9 is an integral part of the signaling cascade leading to EDHF-mediated responses in resistance arteries.

	ACKNOWLEDGMENTS

 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 553/B1 and B2) and Institut de Recherches Internationales Servier. It contains data from the doctoral thesis of S.P.

	FOOTNOTES

 
Received for publication March 26, 1999. Revised for publication September 20, 1999.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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