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Transfection of CYP4A1 cDNA decreases diameter and increases responsiveness of gracilis muscle arterioles to constrictor stimuli

¹Department of Pharmacology, New York Medical College, Valhalla, New York 10595; and ²Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Submitted 3 July 2003 ; accepted in final form 3 May 2004

	ABSTRACT

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Cytochrome P-450–4A1 (CYP4A1) is an -hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4A1. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 ± 3 µm) was surpassed (P < 0.05) by that of vessels transfected with control plasmid (97 ± 4 µm). Treatment with a CYP4A inhibitor (N-methylsulfonyl-12,12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40–100 mmHg elicited a more intense (P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product.

myogenic vasoconstriction; 20-hydroxyeicosatetraenoic acid; cytochrome P-450 oxygenases; vascular reactivity

Recently, we reported that transfection of small renal arterial vessels with an expression plasmid containing the cDNA of CYP4A1, an arachidonic acid -hydroxylase that catalyzes the synthesis of 20-HETE with greater efficiency than other CYP4A isoforms (16), increases the vascular expression of CYP4A protein along with the production of 20-HETE (14). In the present study, we used this experimental approach to investigate the vasomotor consequences of increased CYP4A1 expression in rat gracilis muscle arterioles. We compared isolated rat gracilis muscle arterioles transfected with an expression plasmid with and without CYP4A1 cDNA in terms of CYP4A1 protein expression, internal diameter at various levels of transmural pressure, and constrictor responsiveness to phenylephrine. The vessels also were compared in terms of their vasomotor responses to agents that interfere with the synthesis or actions of 20-HETE.

	MATERIALS AND METHODS

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Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (250–275 g, Charles River, Wilmington, DE) were anesthetized with pentobarbital sodium (60 mg/kg ip). The gracilis anticus muscle was excised and placed on a dish filled with ice-cold Krebs buffer composed of (in mmol/l) 118.5 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 25.0 NaHCO₃, and 11.1 dextrose, and first-order gracilis muscle arterioles were immediately dissected for use in various protocols.

Transfection of gracilis muscle arterioles. Full-length CYP4A1 cDNA (16) was cloned into the expression vector pcDNA3.1(+) (Invitrogen; Carlbad, CA). The expression plasmid containing CYP4A1 cDNA (pcDNA3.1–4A1) and the control plasmid (pcDNA3.1) were encapsulated into liposomes (DOTAP Liposomal Transfection Reagent, Boehringer Mannheim; Indianapolis, IN). First-order gracilis muscle arterioles were placed on culture dishes (35 mm) filled with tissue culture medium (Dulbecco's modified Eagle's medium with 10% Nu-serum, 100 µg/ml streptomycin, and 100 µg/ml penicillin) containing 20 µg/ml of liposome-encapsulated pcDNA3.1–4A1 or pcDNA3.1. The vessels were maintained in organ culture for 18 h in a humidified incubator (95% air-5% CO₂) at 37°C. At the end of the culture period, the vessels were used for assessment of CYP4A protein expression and for examination of vasomotor function.

Assessment of CYP4A protein. Samples consisting of six to eight gracilis muscle arterioles, maintained in organ culture as described above, were homogenized, the homogenate was centrifuged, and the 10,000-g supernatant (25 µg of protein) was analyzed for CYP4A protein by immunoblotting with the use of a goat anti-rat CYP4A1 polyclonal antibody (Gentest; Woburn, MA) as previously described (14). Immunoreactive proteins were detected by using the ECL Plus detection system (Amersham; Arlington Heights, IL).

Immunohistochemistry. Gracilis muscle arterioles were fixed in 4% formaldehyde in phosphate-buffered saline (PBS). After being washed with PBS, the vessels were dehydrated in ethanol, embedded in Tissue-Tek OCT (Sakura; Torrance, CA), and cut into 5-µm sections, which were placed onto polylysine-coated slides and incubated for 2 h with blocking solution containing 3% nonimmune goat IgG and 0.25% Triton X-100. The slides were then washed with PBS, incubated with goat anti-rat CYP4A1 polyclonal antibody (1:1,000 dilution; Gentest, Woburn, MA), which cross-reacts with other members of the CYP4A family, followed by further incubation with rabbit anti-goat IgG (1:1,000 dilution) labeled with Alexa Fluor 594 (Molecular Probes; Eugene, OR). Immunostaining was visualized by confocal microscopy.

Evaluation of vasomotor function. Segments (1–2 mm length) of first-order gracilis muscle arterioles, freshly isolated or maintained in organ culture as described above, were mounted between two micropipettes in the chamber (1 ml) of a pressure-myograph (Living System Instrumentation; Burlington, VT) filled with Krebs buffer gassed with 95% O₂-5% CO₂, which was exchanged at a rate of 1 ml/min (21). To measure vascular diameter, the vessel chamber was placed on the stage of a microscope fitted with a video camera (Javelin; Newburgh, NY) linked to a video caliper (Texas A&M; College Station, TX) and a recorder (21). Intraluminal pressure was increased slowly to 40 or 80 mmHg by using a pressure servocontroller, and this level of pressure was maintained throughout the study unless indicated otherwise. Experiments were conducted after a 60-min equilibration period. The internal diameter of vessels was monitored continuously before and during experimental observations. Pharmacological agents were included into the Krebs buffer flowing into the myograph chamber. The following protocols were implemented.

In protocol 1, gracilis muscle arterioles treated with pcDNA3.1–4A1 or with the control plasmid pcDNA3.1, which developed a spontaneous tone while pressurized to 80 mmHg, were contrasted in terms of internal diameter before and during exposure to N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 µmol/l), a selective inhibitor of CYP4A isoenzymes (16), or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE; 1 µmol/l), an agent reported to block the vascular actions of 20-HETE (1). Arterioles treated with pcDNA3.1 or with pcDNA3.1–4A1 also were contrasted in terms of internal diameter during superfusion with calcium-free Krebs buffer containing 1 mmol/l EGTA.

In protocol 2, the intraluminal pressure-internal diameter relationship in gracilis muscle arterioles treated with pcDNA3.1–4A1 was compared with that in arterioles treated with the control plasmid pcDNA3.1. The comparisons were conducted in preparations superfused with Krebs buffer containing and not containing DDMS (30 µmol/l) or 20-HEDE (1 µmol/l). The pressure-diameter relationship in arterioles treated with pcDNA3.1–4A1 and exposed to DDMS (30 µmol/l) also was examined during superfusion with buffer containing and not containing 20-HETE (1–10 µmol/l). Additional experiments conducted in freshly isolated gracilis muscle arterioles compared the pressure-diameter relationship in preparations superfused with Krebs buffer only, buffer containing DDMS (30 µmol/l) or 20-HEDE (1 µmol/l), and buffer containing 20-HETE (1 µmol/l) along with DDMS or 20-HEDE. The intraluminal pressure-internal diameter relationship was studied as previously described (21). After equilibration at 80 mmHg, the pressure was decreased to 0 mmHg and, after 10 min, it was increased in 20-mmHg steps until it reached 100 mmHg. The pressure was maintained for 5–10 min at each pressure step so that the vessels could reach a steady-state diameter. Before an experiment was concluded, the vascular preparation was superfused with calcium-free Krebs buffer containing 1 mmol/l EGTA, and the pressure-diameter relationship was examined again to obtain the passive diameter of the vessels at each level of intraluminal pressure. The internal diameter during superfusion of the arterioles with calcium-containing buffer (absolute diameter) and with calcium-free buffer (passive diameter) are expressed in micrometers. The normalized diameter refers to the absolute diameter expressed as a percentage of the passive diameter.

In protocol 3, gracilis muscle arterioles transfected with pcDNA3.1–4A1 or with the control plasmid pcDNA3.1 were pressurized to 40 mmHg and contrasted in terms of constrictor responsiveness to the cumulative addition of phenylephrine (10^–9–10^–5 mol/l) to the superfusion buffer; the experiments were conducted in preparations superfused with Krebs buffer alone, buffer containing DDMS (30 µmol/l), and buffer containing both DDMS and 20-HETE (1 µmol/l).

Data analysis. Results are expressed as means ± SE. Data were analyzed by unpaired Student's t-test or by two-way ANOVA, followed by the Newman-Keuls post hoc test. The null hypothesis was rejected at P < 0.05.

	RESULTS

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Figure 1 illustrates the results of Western blot analysis of proteins in isolated rat gracilis muscle arterioles maintained in organ culture in media supplemented with an expression plasmid containing pcDNA3.1–4A1 or the control plasmid pcDNA3.1. The expression of CYP4A protein(s) in vessels treated with pcDNA3.1–4A1 greatly exceeds that in vessels treated with the control plasmid pcDNA3.1. Figure 2 shows sections of gracilis muscle arterioles transfected with pcDNA3.1 (Fig. 2A) or pcDNA3.1–4A1 (Fig. 2B) displayed an immunofluorescent signal indicative of the expression of CYP4A protein. In arterioles transfected with the control plasmid, the immunofluorescent signal was particularly intense in the intima, whereas in arterioles transfected with pcDNA3.1–4A1, the signal was intense throughout the vessel wall. Little or no immunofluorensce was detected in the control, the sections of arterioles transfected with pcDNA3.1 (Fig. 2C), or pcDNA3.1–4A1 (Fig. 2D) in which incubation with CYP4A antibody was omitted from the immunostaining protocol.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Assessment of cytochrome P-450–4A (CYP4A) expression by immunoblotting in first-order gracilis muscle arterioles maintained in organ culture for 18 h in media supplemented with an expression plasmid containing pcDNA3.1–4A1 or with the control plasmid pcDNA3.1. Representative immunoblots of CYP4A (top) and bar graph (bottom) depicting densitometric analysis (means ± SE of 4 experiments) of CYP4A bands in relation to -actin are shown. *P < 0.05.

View larger version (137K): [in this window] [in a new window]   Fig. 2. Immunostaining of CYP4A protein in sections of gracilis muscle arterioles transfected with pcDNA3.1 (A and C) or pcDNA3.1–4A1 (B and D). CYP4A antibody was omitted from the immunostaining protocol in sections shown in C and D. Magnification is x40 in all panels.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Internal diameter (ID) of pressurized (80 mmHg) first-order gracilis muscle arterioles transfected with pcDNA3.1 or pcDNA3.1–4A1 plasmids, before and after exposure to N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS, 30 µmol/l; A) or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE; 1 µmol/l; B). Data are means ± SE. *P < 0.05 relative to values before treatment with DDMS or 20-HEDE. Numbers in parentheses indicate the number of rats.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effect of stepwise increments in intraluminal pressure on the absolute (A) and the normalized ID (B) of first-order gracilis muscle arterioles transfected with pcDNA3.1 or pcDNA3.1–4A1 plasmids. Data are means ± SE. *P < 0.05 relative to corresponding data in pcDNA3.1-treated vessels. Numbers in parentheses indicate the number of rats.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Effect of DDMS (30 µmol/l) and 20-HEDE (1 µmol/l) on the intraluminal pressure-ID relationship in first-order gracilis muscle arterioles transfected with pcDNA3.1–4A1 plasmids. Data are means ± SE. *P < 0.05 relative to corresponding values in control vessels. Numbers in parentheses indicate the number of rats.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effect of 20-HETE on the intraluminal pressure-ID relationship in first-order gracilis muscle arterioles transfected with pcDNA3.1–4A1 and pretreated with DDMS (30 µmol/l). Data are means ± SE. *P < 0.05 relative to corresponding control values. Number in parentheses indicate the number of rats.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Effect of stepwise increments of intraluminal pressure on the absolute and the normalized ID of freshly isolated first-order gracilis muscle arterioles. Vascular preparation were superfused with control Krebs buffer only, buffer containing DDMS (30 µmol/l) or 20-HEDE (1 µmol/l), or buffer containing 20-HETE (1 µmol/l) along with DDMS (30 µmol/l) or 20-HEDE (1 µmol/l). Data are means ± SE. *P < 0.05 relative to corresponding data in vessels superfused with Krebs buffer only. Number in parentheses indicate the number of rats.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Concentration-dependent constrictor responses to phenylephrine in gracilis muscle arterioles transfected with pcDNA3.1 (A) or pcDNA3.1–4A1 (B) and superfused with control Krebs buffer only, buffer containing DDMS (30 µmol/l), or buffer containing both DDMS (30 µmol/l) and 20-HETE (1 µmol/l). Data are means ± SE. *P < 0.05 relative to corresponding data in vessels superfused with Krebs buffer only. Number in parentheses indicate the number of rats.

	DISCUSSION

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The major finding of our study is that gracilis muscle arterioles transfected with CYP4A1 cDNA differ functionally from nontransfected vessels. That the internal diameter of pcDNA3.1–4A1-treated arterioles, pressurized to 80 mmHg, is about 70% of the internal diameter of pcDNA3.1-treated arterioles at the same pressure level implies that CYP4A1 expression is conducive to vasoconstriction. That the CYP4A inhibitor DDMS elicits robust dilation of pressurized arterioles transfected with pcDNA3.1–4A1 suggests prominent contribution of a product of fatty acid metabolism by CYP4A1 to the implementation of constrictor tone in these vessels. It is plausible that the product in question is arachidonic acid-derived 20-HETE, because 20-HEDE (1), a putative antagonist of 20-HETE, also was effective in eliciting robust dilation of pressurized gracilis muscle arterioles transfected with pcDNA3.1–4A1. However, CYP4A1 also was reported to catalyze hydroxylation of fatty acids other than arachidonic acid (viz., palmitic, myristic, and oleic acids) (3, 12), raising the possibility that one or more of the resulting products contributes to the constrictor tone displayed by gracilis muscle arterioles overexpressing CYP4A1. Involvement of CYP4A-derived products in the implementation of constrictor tone in control arterioles transfected with pcDNA3.1 appears to be minimal because little or no dilatory response ensues when such vessels are exposed to DDMS or 20-HEDE. According to our study, CYP4A1 protein expression is diffusely distributed throughout the wall of arterioles treated with pcDNA3.1–4A1, whereas it is somewhat circumscribed to the intima of arterioles treated with pcDNA3.1. It is then conceivable that differences in the intensity of the constrictor tone displayed by these vessels reflect not only differences in the quantity of CYP4A products generated but also in the site of generation, which, in turn, may impact on the number of smooth muscle cells responding to such products.

It is well documented that pressurization of gracilis muscle arterioles promotes development of myogenic constrictor tone (21). 20-HETE of vascular origin is believed to upregulate the expression of myogenic behavior in gracilis muscle arterioles (6) and in small resistance arteries of the mesenteric circulation (20), the kidney (13), and the brain (8). In agreement with this notion, we found that treatment with DDMS or 20-HEDE, which respectively interferes with the synthesis and action of 20-HETE, attenuates the reduction of internal diameter caused by increments of intraluminal pressure in freshly isolated gracilis muscle arterioles. Hence, it is conceivable that the increased vasoconstriction displayed by pressurized gracilis arterioles treated with pcDNA3.1–4A1 is due to magnification of myogenic tone by a CYP4A1-derived product, including 20-HETE. This concept is in keeping with observations that pressure-induced reduction of internal diameter over the pressure range of 40–100 mmHg is significantly enhanced in gracilis muscle arterioles treated with pcDNA3.1–4A1, and that inclusion of DDMS or 20-HEDE into the superfusion buffer brings down the expression of pressure-induced vasoconstriction in such vessels to the level observed in preparations treated with pcDNA3.1. Also supportive of this concept is the finding that exogenous 20-HETE offsets the inhibitory effect of the 20-HETE synthesis inhibitor DDMS on pressure-induced constriction of arterioles overexpressing CYP4A-1.

In agreement with the results of a previous study in rat renal interlobar arteries (14), we found the gracilis muscle arterioles transfected with pcDNA3.1–4A1 are more sensitive to phenylephrine than arterioles transfected with the control plasmid pcDNA3.1. The sensitizing influence of treatment with pcDNA3.1–4A1 is attributable to increased vascular synthesis of a CYP4A1 product, because when the experiments are conducted in the presence of DDMS, pcDNA3.1–4A1-treated arterioles are no longer more sensitive to phenylephrine than arterioles treated with the control plasmid. Hence, enhancement of constrictor responsiveness in gracilis muscle arterioles overexpressing CYP4A1 is not limited to myogenic stimuli but extends to phenylephrine as well.

The present study adds to a large body of evidence that a CYP4A-derived product(s) of vascular origin serves as a facilitatory modulator of microvascular reactivity to diverse constrictor stimuli, including constrictor agonists (22), increased oxygen tension (9–11, 15, 17, 19, 22, 23), and increments of transmural pressure (6, 8, 14). CYP4A-derived 20-HETE is regarded as a prime candidate for playing such a role as production of 20-HETE by small arterial vessels is increased in experimental settings that bring about vasoconstriction, e.g., elevation of transmural pressure (8), augmentation of oxygen tension (11), exposure to angiotensin II (4). Moreover, the known abilities of 20-HETE to inhibit Ca²⁺-activated K⁺ channels in vascular smooth muscle (18, 23), to enhance the conductance of Ca²⁺ channels (8, 9, 18), and to activate Rho kinase (17) are all expected to promote amplification of vasoconstrictor responsiveness. It is not known whether 20-HETE shares with CYP4A products arising from hydroxylation of fatty acids other than arachidonic acid the ability to promote vasoconstrictor responsiveness. If it does, such products may be considered along with 20-HETE potential contributors to the functional manifestations of vascular CYP4A product generation.

Studies by other investigators have linked vascular CYP4A product generation to physiological mechanisms of microcirculatory adjustment in the face of elevation of perfusion pressure (5–8) and oxygen tension (11). In addition, augmentation of vasoconstrictor responsiveness in spontaneously hypertensive rats (22), Dahl salt-sensitive hypertensive rats (7), and rats with reduced renal mass hypertension (5) were shown to rely on a mechanism involving CYP4A oxygenases, which is expressed more prominently in hypertensive than in normotensive rats (7, 22).

In summary, this study demonstrates overexpression of CYP4A protein in rat gracilis muscle arterioles maintained in culture for 18 h in media that includes an expression plasmid containing CYP4A1 cDNA. The internal diameter of pressurized arterioles transfected with pcDNA3.1–4A1 was greatly exceeded by that of arterioles transfected with the control plasmid. The constrictor influence of CYP4A overexpression is attributable to facilitation of myogenic vasoconstriction by a CYP4A-derived product.

	GRANTS

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The work was supported by National Institutes of Health Grants HL-34300, HL-18579, and DK-38226.

	ACKNOWLEDGMENTS

 
We thank Jennifer Brown for assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: F. Zhang, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: Fan_Zhang{at}nymc.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

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This Article Abstract Full Text (PDF) All Versions of this Article: 287/3/H1089    most recent 00627.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Zhang, F. Articles by Nasjletti, A. Search for Related Content PubMed PubMed Citation Articles by Zhang, F. Articles by Nasjletti, A.