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(Hypertension. 1998;31:242.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Cytochrome P-450 Metabolites Mediate Norepinephrine-Induced Mitogenic Signaling

Mohammed R. Uddin; Mubarack M. Muthalif; Nour A. Karzoun; Ibrahim F. Benter; Kafait U. Malik

From the Department of Pharmacology, College of Medicine, The University of Tennessee Center for Health Sciences (M.M.M. and K.U.M.), Southern College of Optometry (N.A.K. and I.F.B.), and LeMoyne Owen College (M.R.U.), Memphis, Tenn.

Correspondence to Kafait U. Malik, DSc, PhD, Professor, Department of Pharmacology, College of Medicine. The University of Tennessee, The Health Science Center, Memphis, TN 38163. E-mail kmalik{at}utmem1.utmem.edu

	Abstract

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Norepinephrine (NE) stimulates release of arachidonic acid (AA) from tissue lipids in blood vessels, which is metabolized via cyclooxygenase, lipoxygenase (LO), and cytochrome P-450 (CYP-450) pathways to biologically active products. Moreover, NE and AA have been shown to stimulate proliferation of vascular smooth muscle cells (VSMCs) of rat aorta. The purpose of this study was to determine the possible contribution of AA and its metabolites to NE-induced mitogenesis in VSMCs of rat aorta and the underlying mechanism of their actions. NE (0.1 to 10 µmol/L) increased DNA synthesis as measured by [³H]thymidine incorporation in VSMCs, and this effect was attenuated by inhibitors of CYP-450 (17-octadecynoic acid, 5 µmol/L; 12-diabromododec-11-enoic acid, 10 µmol/L; and dibromo-dodecenyl-methylsulfimide, 10 µmol/L) and by the LO inhibitor (baicalein, 20 µmol/L), but not by the cyclooxygenase inhibitor (indomethacin, 5 µmol/L). CYP-450 and LO metabolites of AA, 20-hydroxyeicosatetraenoic acid (HETE) (0.1 to fimide, 10 µmol/L) and by the LO inhibitor (baicalein, 20 µmol/L), but not by the cyclooxygenase inhibitor (indomethacin, 5 µmol/L). CYP-450 and LO metabolites of AA, 20-hydroxyeicosatetraenoic acid (HETE) (0.1 to 0.5 µmol/L) and 12(S)-HETE, respectively, increased [³H]thymidine incorporation in VSMCs. Both NE and 20-HETE increased mitogen activated protein (MAP) kinase activity as measured by the in-gel kinase assay. The inhibitor of MAP kinase kinase, PD-98059 (50 µmol/L), attenuated NE as well as 20-HETE induced [³H]thymidine incorporation and MAP kinase activation in VSMCs. These data suggest that products of AA formed via CYP-450, most likely 20-HETE, and via LO mediate NE induced mitogenesis in VSMCs.

Key Words: vascular smooth muscle cells • mitogenesis • 20-hydroxyeicosatetraenoic acid • norepinephrine • MAP kinase • proliferation

Abbreviations: AA = arachidonic acid • BACL = baicalein • COX = cyclooxygenase • cPLA₂ = cytosolic phospholipase A₂ • CYP-450 = cytochrome P-450 • DBDD = 12-dibromododec-11-enoic acid • DDMS = dibromo-dodecenyl-methylsulfimide • ERK = extracellular regulated kinase • FBS = fetal bovine serum • HETE = hydroxyeicosatetraenoic acid • IND = indomethacin; • LO = lipoxygenase • MAFP = methyl arachidonyl fluoro phosphonate; MAP kinase mitogen activated protein kinase • MEK = MAP kinase kinase; MBP, myelin basic protein • NE = norepinephrine • 17-ODYA = 17-octadecynoic acid; TBS, tris-buffer saline • VSMC = vascular smooth muscle cell

	Introduction

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Sympathetic nerve stimulation or catecholamines promote hypertrophy and hyperplasia of smooth muscle cells in blood vessels, which has been implicated in the pathogenesis of hypertension and atherosclerosis.^1–6 NE stimulates proliferation of subconfluent VSMCs^7,8 and hypertrophy without mitogenesis in quiescent confluent VSMCs.^9–11 NE-induced VSMC hyperplasia is mediated by activation of ₁-adrenergic receptors.^12,13 Recent studies have shown that _1B and/or _1D subtypes of ₁-adrenergic receptors are involved in rat VSMC hyperplasia and hypertrophy.^9,14,15

Activation of -adrenergic receptors with NE has also been reported to stimulate AA release in a Ca²⁺/calmodulin-dependent manner via activation of cPLA₂.^16,17 Recently, we have shown that NE stimulates cPLA₂ and AA release by activating Ca²⁺/calmodulin-dependent kinase II.¹⁷ AA and the products of its metabolism have been shown to stimulate growth in many cell types including VSMCs.^18–22 Moreover AA and LO metabolites stimulate MAP kinase activity.²² These observations and the recent finding that NE-induced hyperplasia is dependent on MAP kinase activation^11,14 have led us to hypothesize that NE-induced VSMC proliferation is mediated by AA and/or its metabolites via MEK activation. To test this hypothesis, we have investigated the effect of NE and AA on rat aortic smooth muscle cell proliferation, measured by [³H]thymidine incorporation, in the presence of inhibitors of cPLA₂- or AA-metabolizing enzymes, COX, LO, and CYP-450 or MEK.

	Methods

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Materials
Hanks’ balanced salt solution, M-199, phosphate buffered saline, bovine serum albumin, dithiothreitol, EGTA, NE, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and myelin basic protein were purchased from Sigma; leupeptin and aprotinin were from Calbiochem; [methyl-³H]thymidine (20 Ci/mmol) was obtained from Du Pont-NEN; and [-³²P]ATP (3000Ci/mmol) was obtained from Amersham.

MEK inhibitor PD-98059²³ was obtained from New England Biolabs; cPLA₂ inhibitor MAFP²⁴ and 12(S)-, and 20-HETE were from Cayman Chemicals. COX inhibitor IND²⁵ CYP-450 inhibitor 17-ODYA²⁶ and LO inhibitor BACL²⁷ were purchased from Biomol. DBDD²⁸ and DDMS²⁹ were synthesized by Dr. John R. Falck (University of Texas Southwestern Medical Center, Dallas, Tx) and kindly provided by Dr. Alberto Nasjletti (New York Medical College, Valhalla, NY) and Dr. Richard Roman (Medical College of Wisconsin, Milwaukee, Wis), respectively. All inhibitors were dissolved in dimethyl sulfoxide and further diluted with M-199 for experiments.

Preparation of VSMCs
The following protocol was reviewed and approved by our Institution Animal Care and Use Committee and conforms with the Guide for the Care and Use of Laboratory Animals (National Institute of Health). Sprague-Dawley rats (250 to 350g; Charles River, Wilmington, Mass) were anesthetized with 30 mg/kg pentobarbital (Abbott Laboratories). The thoracic aortae was rapidly removed, and VSMCs were isolated and cultured as previously described.²⁹

Measurement of DNA Synthesis via [³H]Thymidine Incorporation
Incorporation of [³H]thymidine into DNA was measured in aortic smooth muscle cells isolated and cultured as described.³⁰ Subconfluent cells from fifth through ninth passages were incubated with 0.05% FBS containing M-199 for 48 hours to induce mitogenic quiescence. Cells were incubated with NE (0.01 to 1 µmol/L) or its vehicle for 48 hours and 0.5 µCi/mL [³H]thymidine was added to the cultures in each well during the last 24 hours of incubation period. This time of incubation with NE and [³H]thymidine resulted in maximal [³H]thymidine incorporation in VSMCs. To investigate the contribution of AA metabolites to the action of NE on DNA synthesis, quiescent cells were preincubated with IND,²⁵ BACL,²⁷ 17-ODYA,²⁶ and DBDD and DDMS (relatively selective for /-1 hydroxylase than epoxygenase)^28,29 for 30 minutes or PD-98059²³ for 4 hours, inhibitors of COX, LO, and CYP-450 or MEK (5–50 µmol/L), respectively, or their vehicles and then exposed to NE (1 µmol/L) for 48 hours in the presence of the above inhibitors. The effect of exogenous AA on [³H]thymidine incorporation in VSMCs was also studied in the presence of the inhibitors of COX, LO, and CYP-450. These concentrations have been reported to be effective in other cell systems.^25–29 In all experiments 100 µmol/L ascorbate was present with NE to prevent its degradation.³¹ In all cells [³H]thymidine incorporation was normalized for protein content and is expressed as counts per minute per well.

In-Gel MAP Kinase Assay
Control or NE-treated VSMCs were rinsed with ice-cold phosphate-buffered saline (pH 7.4) and immediately scraped into 200 µL/dish of 50 mmol/L ß-glycerophosphate buffer, pH 7.4, containing 1 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, 0.01 mmol/L sodium orthovanadate, and 1 mmol/L dithiothreitol. MAP kinase activity within the supernatant was analyzed using the in-gel kinase method as described.³²

Data Analysis
The results are expressed as means±SE and were analyzed by one-way ANOVA. The Newman-Keuls multiple range test was applied to determine the difference among multiple groups, and the unpaired Student’s t test was used to determine the difference between two groups. The null hypothesis was rejected at P<.05.

	Results

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NE-Stimulated [³H]Thymidine Incorporation in VSMCs Is Mediated by AA Release
To determine the effect of NE on VSMC growth, monolayers of rat aortic VSMCs, made quiescent for 48 hours, were exposed to NE or its vehicle for 48 hours. NE increased [³H]thymidine incorporation in VSMCs in a concentration-dependent manner (0.01 to 1 µmol/L). A further increase in NE concentration (10 µmol/L) diminished its effect on [³H]thymidine incorporation in VSMCs (Fig 1). It was demonstrated previously that NE stimulates Ca²⁺/calmodulin-dependent kinase II, leading to activation of MAP kinase and cPLA₂, which results in release of AA.¹⁷ To determine whether AA is involved in NE-stimulated [³H]thymidine uptake, the cells were preincubated with the cPLA₂ inhibitor MAFP. NE-induced [³H]thymidine uptake was attenuated by MAFP (Fig 2). These data suggest that release of AA is required for NE-stimulated [³H]thymidine uptake in VSMCs.

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of NE on [3H]-thymidine incorporation in rat VSMCs. Quiescent VSMCs grown in 48-well plates in arresting medium (0.05% FBS) were treated with NE (0.01 to 10 µmol/L) or 1% FBS for 48 hours. In the last 24 hours cells were labeled with [3H]thymidine (0.05 µCi/mL), and incorporation into DNA was determined as described in "Methods." Results are mean±SE; n=5 to 6. *Value significantly different from vehicle (VEH) (M-199 + ascorbate) (P<.05).

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of the cPLA2 inhibitor MAFP on NE-induced [3H]thymidine incorporation in VSMCs. Quiescent cells were pre-treated for 4 hours with 40 µmol/L MAFP. Thymidine incorporation was measured as described in "Methods" and is presented as cpm per well. Results are mean±SE; n=5 to 6. *Value significantly different from vehicle (VEH) of NE; Value different from that obtained with VEH of MAFP.

CYP-450 and LO But Not COX Metabolites of AA Mediate NE-Stimulated [³H]Thymidine Uptake
Three major enzyme systems—COX, LO, and NADPH-dependent CYP-450 monooxygenase—metabolize AA into products that have prominent biological actions, including modulation and mediation of the actions of vasoactive hormones.^33,34 AA metabolites formed through COX, LO, or CYP-450 pathways may be a prerequisite for NE to stimulate proliferation of VSMCs. To test this possibility, the effect of inhibitors of COX, LO, or CYP-450 on NE-stimulated [³H]thymidine incorporation was studied. Cells were preincubated with inhibitors of COX (IND), LO (BACL), or CYP-450 (17-ODYA), or /-1 hydroxylase (DBDD and DDMS) and then exposed to NE for 48 hours in the presence of inhibitors. As shown in Fig 3, the CYP-450 and LO inhibitors, but not the COX inhibitor, attenuated [³H]thymidine incorporation. Combined treatment with 17-ODYA and BACL decreased NE-induced [³H]thymidine uptake to a much greater degree than either of these agents alone. The contribution of /-1 hydroxylation products of AA to the proliferative action of VSMCs by NE was studied after the endogenous production of HETEs was blocked with DBDD and DDMS. Treatment of VSMC with DBDD and DDMS attenuated NE induced [³H]thymidine uptake in VSMCs. AA (20 µmol/L) also increased [³H]thymidine incorporation, and this increase was inhibited by 17-ODYA and BACL but not by IND in VSMCs (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 3. Effect of inhibitors of the CYP-450, LO, and COX on NE-stimulated [3H]thymidine incorporation in VSMCs. Quiescent cells were pretreated with inhibitors of CYP-450 (17-ODYA, 5 µmol/L; DBDD, 10 µmol/L; and DDMS, 10 µmol/L), LO (BACL, 20 µmol/L), and COX (IND, 5 µmol/L) and then stimulated with NE (1 µmol/L) for 48 hours in the presence of the inhibitors. [3H]thymidine incorporation was measured as described in "Methods" and is presented as cpm per well. Results are mean±SE; n=5 to 6. *Value significantly different from that obtained with vehicle (VEH); Value significantly different from that obtained with VEH of the inhibitors; Value significantly different from that obtained with BACL or 17-ODYA alone (P<.05).

None of the inhibitors was cytotoxic to the cells at the concentrations used, as measured by exclusion of trypan blue (0.4%); more than 95% of the cells were impermeable to this dye (data not shown). These results suggest that one or more AA metabolites generated via CYP-450 and LO is required for NE-stimulated [³H]thymidine uptake in VSMCs.

20- and 12(S)-HETE Stimulated VSMC Proliferation
Attenuation of NE-stimulated [³H]thymidine uptake by 17-ODYA, which inhibits CYP-450, and by DBDD and DDMS, which selectively inhibit /-1 hydroxylation of AA to HETEs including 20-HETE formation, and BACL, which inhibits 12-LO, suggests that 20- and 12(S)-HETE might be the mediators of NE-induced proliferation of VSMCs. As shown in Fig 4, 20-HETE increased [³H]thymidine incorporation into VSMCs in a concentration-dependent manner. The effect was maximal at 0.5 µmol/L. 12(S)-HETE at 100 and 500 nmol/L also increased [³H]thymidine uptake into VSMCs by 56.6±6.09% and 48.58±15.71% respectively (n=3, P<.05). 12(R)-, and 19-HETE did not alter [³H]thymidine uptake into VSMCs (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of 20-HETE and its vehicle (VEH) on [3H]thymidine incorporation in VSMCs. Cells were arrested with 0.05% FBS and treated with 20-HETE (10 nmol/L to 0.5 µmol/L) or its vehicle (ethanol, 0.5 µL/mL) for 48 hours. In the last 24 hours, [3H]thymidine (0.5 µCi/mL) was added, and incorporation was determined as described in "Methods." Results are expressed as cpm/well and are mean±SE; n=5–6. *Value significantly different from that obtained with vehicle (VEH).

MAP Kinase Mediates NE- and 20-HETE-Stimulated VSMC Proliferation
MAP kinases are activated during the transition of cells from the G₀/G₁ to the S phase of the cell cycle.³⁵ Therefore, the contribution of MAP kinase to NE- and 20-HETE-stimulated [³H]thymidine incorporation was studied by pretreating cells with PD-98059, a MEK inhibitor. PD-98059 in a concentration-dependent manner attenuated NE- and 20-HETE-stimulated [³H]thymidine incorporation (Fig 5). PD-98059 (20 µmol/L) inhibited the FBS (1%)-induced increase in [³H]thymidine uptake in VSMCs by 50% (n=2). This compound did not alter the basal levels of [³H]thymidine uptake in VSMCs. Both NE and 20-HETE increased ERK1 and ERK2 MAP kinase activity as indicated by phosphorylation of MBP (Fig 6A). The increase in the activity of both ERK1 and ERK2 elicited by NE and 20-HETE was inhibited by PD-98059 (Fig 6B). Therefore, it appears that (MAP) kinase mediates NE-, 20-HETE-, and 12(S)-HETE-stimulated proliferation in VSMCs.

View larger version (45K): [in this window] [in a new window]   Figure 5. Effect of MEK inhibitor PD-98059 (5 to 50 µmol/L) on NE (upper panel)- and 20-HETE (lower panel)-stimulated [3H]thymidine incorporation in VSMCs. Quiescent cells were pretreated for 4 hours with 50 µmol/L PD-98059 and then exposed to NE or to 20-HETE in the presence of this agent. [3H]Thymidine incorporation was measured as described in "Methods" and is presented as cpm per well. Results are mean±SE; n=5 to 6. *Value significantly different from that obtained with vehicle (VEH) of NE; Value significantly different from that obtained with VEH of PD-98059 (P<.05).

View larger version (70K): [in this window] [in a new window]   Figure 6. A, Effect of NE and 20-HETE on MAP kinase activation in VSMCs. Quiescent VSMCs were stimulated with 20-HETE (0.1, 0.25, and 0.5 µmol/L) or NE (0.1, 1, 5, and 10 µmol/L) and for 5 minutes. Cell homogenates (25 µg) were assayed for MAP kinase activity by the in-gel kinase method as described. Arrows indicate 44- and 42-kD bands identified as ERK1 and ERK2. B, Effect of MEK inhibitor PD-98059 (50 µmol/L) on NE- and 20-HETE-stimulated MAP kinase in VSMCs. VSMCs were pretreated with PD-98059 (50 µmol/L) for 4 hours and stimulated with NE (1 µmol/L) and 20-HETE (250 nmol/L) for 5 minutes.

	Discussion

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Recently we have demonstrated that NE-stimulated Ca²⁺/calmodulin-dependent kinase II activates MAP kinase, which in turn increases cPLA₂ activity and releases AA in VSMCs.¹⁷ AA has been shown to increase MAP kinase activity and promotes mitogenesis in rat VSMCs via its conversion by LO into 15-HETE.²² Angiotensin II has also been reported to stimulate MAP kinase and cause mitogenesis via generation of the LO product 12-HETE in VSMCs.²⁰

Our study indicates that 12(S)-HETE increased VSMC proliferation in the rats. The present study indicates that, in addition to LO, the CYP-450, but not COX, metabolites of AA consequent to cPLA₂ activation by NE stimulate proliferation of rat VSMCs via activation of MAP kinase. This conclusion is based on our findings that inhibitors of cPLA₂ (MAFP), CYP-450 (17-ODYA), and LO (BACL), but not COX (IND), attenuated NE-induced proliferation, measured by [³H]thymidine incorporation in the rat VSMCs. That the products of AA generated via CYP-450 and LO, but not COX, promote proliferation of VSMCs was indicated by our finding that exogenous AA, like NE, enhanced [³H]thymidine incorporation into VSMCs and that this was inhibited by 17-ODYA and BACL, but not IND. That NE-induced [³H]thymidine incorporation was attenuated by combined treatment with 17-ODYA and BACL to a greater degree than by either of these agents alone supports our contention that both CYP-450 and LO contribute to NE- and AA-induced proliferation of VSMCs. The major product of AA formed via COX in VSMCs, PGI₂, has been shown to inhibit VSMC proliferation, as indicated by decreased FBS-induced DNA synthesis in cells overexpressing prostaglandin I₂ synthase.³⁶ However, in the present study, the effect of NE-induced VSMC proliferation was independent of prostaglandins. The effect of angiotensin II to stimulate proliferation of VSMCs has also been reported to be not altered by the COX inhibitor.²¹

In addition to its metabolism by COX, AA is metabolized by LO into 5-, 12(S)-, and 15-HETE in VSMCs.³⁷ 12-LO protein and mRNA of the leukocyte type has been shown to be expressed in porcine VSMCs, and 12-LO activity and expression were increased by angiotensin II.²⁰ However, recent studies have shown that AA is also metabolized by NADPH-dependent CYP-450 into eicosatrienoic acids and HETEs, including 20-HETE.³⁸ Moreover, CYP-4504A, which metabolizes AA, has been reported to be present in VSMCs isolated from preglomerular rat renal arterioles.³⁹ These observations, together with our finding that 20-HETE increased [³H]thymidine incorporation in rat VSMCs, suggest that this product of CYP-450 contributes to NE-induced proliferation. That 20-HETE generated in response to NE stimulates VSMC proliferation is also suggested by our finding that the relatively selective inhibitors of /-1 hydroxylation DBDD and DDMS^28,29 attenuated NE-induced proliferation of rat VSMCs. 20-HETE has been reported to stimulate proliferation of proximal tubular cells and also to mediate epidermal growth factor-induced proliferation of these cells.⁴⁰ Because 20-HETE is known to be metabolized into 20-carboxylic-HETE,^34,41 we cannot exclude the possibility that this metabolite mediates the effect of 20-HETE in VSMCs.

The mechanism by which CYP-450 and LO products of AA generated in response to NE stimulate VSMC proliferation is not known. AA metabolites of LO, 12(S)-, and 15-HETE have been shown to increase activity of MAP kinase, which has been implicated in NE-induced proliferation of VSMCs.^21,22 Our finding that 20-HETE increased MAP kinase activity and that the increase in MAP kinase as well as proliferation of VSMCs elicited by NE and 20-HETE were attenuated by PD-98059, an inhibitor of MEK, suggest that the effect of NE on VSMC proliferation is mediated by CYP-450 and LO products of AA through MAP kinase activation via MEK stimulation. Whether other kinase pathways (p38 and JAK/STAT) are also involved in NE- and 20-HETE-induced proliferation of rat VSMCs cannot be excluded. MEK is stimulated by Raf, which in turn can be activated by Ras, and NE has been shown to stimulate Ras in human VSMCs.⁷ Therefore, it is possible that the products of AA generated via CYP-450 and LO stimulate Raf by increasing Ras activity or by a mechanism independent of Ras.⁴² HETE generated by AA metabolism via CYP-450 and LO might activate Ras by hydroxyarachidonylation, allowing its binding to the plasma membrane and subsequent activation. Alternatively, it is possible that HETE activates the MAP kinase pathway probably by increasing influx of Ca²⁺ through activation of specific receptors on the cell membrane.

In conclusion, the present study demonstrates that NE through activation of cPLA₂ results in release of AA; the products of AA generated through CYP-450 and LO, most likely 20-HETE and 12(S)-HETE, respectively, promote proliferation of rat VSMCs by activating MAP kinase.

	Acknowledgments

 
This work was supported by US Public Health Service-National Institutes of Health Grant 19134 from the National Heart, Lung and Blood Institute (to K.U.M.), a Center for Neuroscience fellowship and an American Heart Association Tennessee Affiliate Postdoctoral Fellowship (to M.M.M.), and an American Heart Association Tennessee Affiliate New Investigator Award (to I.F.B.). The authors gratefully acknowledge and appreciate the technical assistance of Angela Glass, Anne Estes, and Jason Harper and thank Dr. Lauren Cagen for editorial comments.

Received September 19, 1997; first decision October 9, 1997; accepted October 24, 1997.

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