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Characterization of arachidonic acid metabolism in Watanabe Heritable Hyperlipidemic (WHHL) and New Zealand White (NZW) rabbit aortas
PROSTAGLANDINS CHARACTERIZATION OF ARACHIDONIC ACID METABOLISM IN WATANABE HERITABLE HYPERLIPIDEMIC (WHHL) AND NEW ZEALAND WHITE (NZW) RABBIT AORTAS Sandra L. Pfister, James M. Schmitz, James T. Willerson, and William B. Campbell Departments of Pharmacology and Cardiology The University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Boulevard Dallas, TX 75235-9041 ABSTRACT
WHHL rabbits develop progressive atherosclerosis. There are no visible signs of the disease at 1 month, however, by 12 months, the formation of aortic plaques is extensive. This study characterized arachidonic acid (AA) metabolism in 1 and 12 month old WHHL and NZW rabbit aortas. Vessels incubated with 14C-AA and A23187 metabolized AA to a number of oxygenated products as identified by high pressure liquid chromatography. The major AA metabolites produced by WHHL and NZW aortas were 6-keto PGFI~, PGE2, 12- and 15-hydroxyeicosatetraenoic acids (HETEs). The structures o~ the HETEs were confirmed by gas chromatography-mass spectrometry. Indomethacin blocked the synthesis of prostaglandins (PGs) but not HETEs whereas ETYA, NDGAor removal of the endothelium attenuated the production of b o t h PGs and HETEs. Measurement of 6-keto PGF~a, 12- and 15-HETE by specific radioimmunoassays indicated that as the rabblts aged and as atherosclerosis progressed, aortas lost the a b i l i t y to synthesize 6-keto PGFla and 15-HETE. Prior to the development of atherosclerosis, 1 month old WHHL aortas produced 70% less 15-HETE than did NZW aortas. Atherosclerotic aortas from 12 month old WHHLs synthesized 60% less 6-keto PGFla during stimulation with AA or A23187 than did 12 month old NZW aortas. We conclude that the development and expression of atherosclerosis in WHHL rabbits impairs the a b i l i t y of aortas to metabolize AA to both PGs and HETEs. INTRODUCTION
Studies have suggested that AA metabolism is altered in vessels obtained from atherosclerotic rabbits (1-4). In rabbits fed a high cholesterol diet to induce atherosclerosis, aortas as well as coronary and mesenteric arteries have a decreased a b i l i t y to synthesize prostacyclin (PGI2) (1-2,4). Platelets obtained from these animals produce increased amounts of thromboxane A2 (TxA~) (3). Based on these results as well as similar results in other specles, including man (5,6), i t is hypothesized that an imbalance between PGI2, a vasodilator and inhibitor of platelet aggregation, and TxA2, a vasoconstrictor and promoter of platelet aggregation, contributes to the expression of atherosclerosis (3,7).
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In addition to the cyclooxygenase pathway, AA is metabolized by lipoxygenase enzymes to form hydroperoxyeicosatetraenoicacids (HPETEs) which are then reduced to the HETEs (8,9). HPETEs (and to a lesser extent, HETEs) have a v a r i e t y of biological actions, including vasoconstriction (10), i n h i b i t i o n of PGI2 synthetase (11,12), migration of aortic smooth muscle cells (13), and i n h i b i t i o n of p l a t e l e t aggregation (14,15). Previous studies have demonstrated that vessels from normal rabbits have the capacity to synthesize HETEs (16-18); however, the production of these compounds has not been well characterized. Likewise, the a b i l i t y of vessels from atherosclerotic rabbits to metabolize AA to HETEs has not been studied. The production of HPETEs and HETEs may be altered in atherosclerosis, and may, for example, be responsible for the decreased production of PGI2 in atherosclerotic rabbit vessels. The WHHL rabbit is an inbred strain of rabbits characterized by a deficiency in functional cell surface receptors for low density lipoproteins. The rabbits develop hypercholesterolemia and severe atherosclerosis (19). No v i s i b l e signs of the disease are seen at one month of age despite elevated serum cholesterol concentrations. By 3 months of age, there is gross evidence of atherosclerosis, and at 12 months of age, the formation of atheromatous plaques and foam cell lesions in the aorta is extensive (20). Import a n t l y , the characteristics of atherosclerosis in the WHHL rabbit more closely resembles human f a m i l i a l hypercholesterolemia than does the d i e t induced atherosclerosis observed in cholesterol-fed rabbits (20). WHHL rabbits are therefore a good model in which to investigate the metabolism of AA in vascular tissue and to determine i f the development of atherosclerosis in WHHL rabbits is accompanied by alterations in AA metabolism. The present study characterizes the production of PGs and HETEs in aortas obtained from i and 12 month old WHHL rabbits and compares this to the production of PGs and HETEs in aortic tissue of age-matched control NZW rabbits.
METHODSAND MATERIALS Experimental Animals Male and female NZW rabbits ( i and 12 months of age) were purchased from Hickory H i l l . Male and female homozygous WHHL rabbits (1 and 12 months of age) were obtained from our colony at Dallas. B o t h groups of animals ate standard laboratory rabbit chow. Rabbits were sacrificed by cervical dislocation, and the thoracic aorta was rapidly removed. The vessels were carefully cleaned of a l l adhering f a t and connective tissue and placed in HEPES buffer (0.01 M HEPES, 0.15 M NaCl, 0.005 M KCI, 0.002 M CaCl2, 0.001 M MgCl2, 0.006 M glucose, pH 7.4). The aortas were cut open l o n g i t u d i n a l l y and then sliced with a razor blade into approximate 2-3 mm. wide segments taking care not to disrupt the endothelium. Incubation of Aortic Tissue with Arachidonic Acid
Strips of vessels from each age group of WHHL and NZW rabbits (100 mg of wet weight) were incubated in 1 ml of HEPES buffer (pH 7.4) at 37°C for 15 minutes with 14C-AA (0.05 NCi, 10-7M) and the calcium ionophore, A23187 (20 NM). Parallel incubations were performed under the same conditions but in the absence of aortic tissue to assess the extent, i f any, of auto-oxidation of AA. In a separate series of experiments, vessels from 1 and 12 month old
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NZW and WHHL rabbits were preincubated for 10 minutes with various i n h i b i t o r s (indomethacin, [I0-5M], 5,8,11,14-eicosatetraynoic acid (ETYA),[3 x 10-5M]~ nordihydroguaiaretic acid (NDGA), [5 x 10-~]) or the endothelium was removeo by gentle ru)bing of the intimal surface with a cotton swab, p r i o r to incubation with 14C-AA and A23187. In another set of experiments designed to i d e n t i f y AA metabolites by gas chromatography-mass spectrometry, aortic tissue from ten NZW and WHHL rabbits (at i and 12 months of age) were incubated for 15 minutes at 37°C in 100 ml of HEPES buffer containing 14C-AA (0.05 ~Ci, 5 x 10-5 ) and A23187.
Isolation and Identification of Arachidonic Acid Metabolites Following incubation, the buffer was removed from the vessels and a c i d i f i e d to pH 3.0 with glacial acetic acid. The AA metabolites were extracted over a Baker C118 octadecylsilica (ODS) column as previously described by Powell (21). The ODS column was washed sequentially with 5 ml ethanol and water. The a c i d i f i e d sample (made 15% [ v o l / v o l ] with respect to ETOH) was then added to the column, and the column washed with 5 ml of 15% ethanol, followed by 5 ml water and f i n a l l y by 5 ml petroleum ether. In some experiments, the HETE fraction was eluted with 10 ml chloroform/petroleum ether (50:50) followed by elution of the PGs with 6 ml ethyl acetate. In other experiments, the HETEs and PGs were eluted together with 6 ml ethyl acetate. Recovery of the PGs and HETEs by this extraction procedure was greater than 95%. The extracted samples were evaporated under a nitrogen stream and stored at -40°C until analysis by high pressure l i q u i d chromatography (HPLC). For reverse-phase (RP) HPLC, a C18 column, (RP-18, 5N, 4.6 x 250 mm, Unimetrics) was used. Two d i f f e r e n t solvent systems were employed. In solvent system 1, solvent A was water and solvent B was a c e t o n i t r i l e containing 0.1% glacial acetic acid. The program was a 40 minute linear gradient at a flow rate of i ml/min from 50% solvent B in A to 100% solvent B. The sample was redissolved and injected in 200 Nl of a c e t o n i t r i l e : w a t e r : acetic acid (50:50:0.01). The eluate was collected in 0.2 ml aliquots and r a d i o a c t i v i t y measured. In solvent system I I , solvent A was water containing 0.025 M phosphoric acid and solvent B was a c e t o n i t r i l e . The program consisted of a 40 minute isocratic phase with 31% B in A, followed by a 20 minute linear gradient to 100% B and 20 minute isocratic phase with 100% B. Flow rate was 1 ml/min. The sample was redissolved and injected in 200 Nl of acetonitrile:water:phosphoric and (31:69:0.01). The eluate was collected in 0.5 ml aliquots and r a d i o a c t i v i t y measured. For normal phase HPLC, a s i l i c a gel column (Ultrasphere, 5N, 4.6 x 250 mm, Beckman) was used. Solvent system I l l consisted of solvent A which was hexane containing 0.1% glacial acetic acid and solvent B was hexane containing 0.1% glacial acetic acid and 2% isopropanol. The program consisted of a 40 minute linear gradient from 25% solvent B in solvent A to 75% solvent B in A. The flow rate was 3 ml/min. The sample was redissolved and injected in hexane:isopropanol:acetic acid (995:5:1). The eluate was collected in 0.6 ml aliquots and r a d i o a c t i v i t y measured.
Gas ChromatographyMass Spectrometry (GC-MS) In order to i d e n t i f y the HETEs from aortic tissue by GC-MS, the extracted metabolites were f i r s t chromatographed on RP-HPLC using solvent system I. The fractions corresponding to the HETEs (#100-130) were c o l l e c t i v e l y pooled, acidified, extracted with cyclohexane/ethyl acetate (50:50) and
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rechromatographed on normal phase HPLC using solvent system I I I . The radioactive peaks corresponding to the retention times of the known HETE standards were collected and derivatized. First, the samples were treated with etheral diazomethane for 30 minutes on ice to form the methyl ester. The hydroxyl groups were then converted to the t r i m e t h y l s i l y l (TMS) ethers by addition of 15 Nl bis-(TMS)-trifluoroacetamide. Sampleswere incubated overnight at 40°C and solvent then removed under argon. GC-MSanalysis was performed with a Finnigan 4021 quadrupole mass spectrometer and a Finnigan INCOS 2000 data system. Ionization of the samples was done by electron impact at 65-70 eV. The column used was a 3% SP-2100-DOH on Supelcoport (6 f t , glass). The flow rate of the carrier gas (N2) was 20-30 ml/min and oven temperature was 220°C.
Radioimmunoassays (RIAs) for PGs and HETES Segments of thoracic aorta (10 mg of wet weight) were obtained from 1 and 12 month old NZW and WHHL rabbits. The vessels were incubated for 30 minutes at 25% in 1 ml of HEPES buffer containing either AA (I0-4M), A23187 (20 NM) or buffer alone. The aortic synthesis of PGI2 was measured in the buffer by RIA of i t s stable metabolite,. 6-keto PGFIa The aortic synthesis of 12-HETE and 15-HETE were measured in the buffer "by specific RIAs. The antibodies for 6-keto PGFIa and 15-HETE were produced in our laboratory in rabbits and the antibody for 12-HETE was supplied by Dr. Levine (Brandeis University). The RIA method used to measure the compounds was similar to that previously described (22). The sensitivity of the assays is less than 5 pg for 6-keto PGFIa, 12-HETE and 15-HETE. Cross-reactivity of the various antisera with known AA metabolites is less than 0.1%. Materials [14C (uniformly labeled)]-AA; (3H)-I2(S)-HETE, (3H)-I5(S)-HETE were obtained from New England Nuclear; HPLC grade solvents were from Baker; (3H)-6-keto PGFla was from Amersham; 12(S)-HETE, 15(S)-HETE and 5(S)-HETE were from BioMol; HEPES, A23187, AA, NDGA and indomethacin were from Sigma; l i q u i d s c i n t i l l a t i o n counters were from Beckman and Packard Instruments; budget solve s c i n t i l l a t i o n f l u i d was from Research Products, Inc.; 6-keto PGFI~, PGE2, TxBo, PGF~a, PGD~ were from Upjohn Co." ETYA was a g i f t from HoffmanLaRoche; bls(TMS~ trifluoracetamide was from Supelco, Inc. Statistical Analysis The RIA data were expressed as means ± SEM. Statistical evaluation of the data was performed by using Student's "t" test. RESULTS Arachidonic Acid Metabolism by Aortas of WHHL and NZW Rabbits Gross examination of aortas from I and 12 month old NZW and 1 month old WHHL rabbits appeared normal. Aortas from 12 month old WHHL rabbits showed visible f a t t y streaks and raised plaque areas. Incubation of aortas from various age groups of WHHL and NZW rabbits with 14C-AA and A23187 resulted in the metabolism of AA to a number of oxygenated products as identified by RP-HPLC using solvent system I (Figure i ) . Aortas from 1 month old WHHL and NZW rabbits produced radioactive peaks which corresponded to the elution
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times of the PG metabolites, dihydroxyeicosatetraenoic acids (diHETEs) or dihydroxyeicosatrienoic acids (DHETs), HETEs and AA. As the rabbits aged, the aortas appeared to lose the a b i l i t y to metabolize AA. As shown in Figure i , decreased radioactivity was observed in the regions corresponding to the PG metabolites and HETEs, and no radioactive peaks were evident in the diHETE-DHET region. Although the pattern of metabolites was similar between WHHL and NZW rabbits, the amount of radioactivity detected in incubations of WHHL aortas was less than that detected in incubations of NZW aortas suggesting a decreased metabolism of AA in WHHL aortas. When HEPES buffer containing 14C-AA and A23187 was incubated without aortic tissue, extracted and subjected to HPLC, no AA metabolites were detected (data not shown).
NZW
~.
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12 month 50
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FRACTION Figure 1: Metabolism of 14C-AA by aortas of 1 and 12 month old NZW and WHHL rabbits. Migration of known standard eicosanoids are shown above. These standards indicate the relative retention times of the known compounds and are not meant to identify the corresponding radioactive peaks.
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To further identify the PGs produced by aortas of WHHL and NZW rabbits, extracted samples were analyzed by RP-HPLC using solvent system I I (Figure 2). This system separates the cyclooxygenase metabolites of AA, whereas the more non-polar lipoxygenase products are unresolved. The retention time of the major cyclooxygenase radioactive peak in aortas from all rabbits corresponded exactly to the retention time of 6-keto PGFla. The smaller peak corresponded to the retention time of PGE2.
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Figure 2: Metabolism of 14C-AA by aortas of 1 and 12 month old NZW and WHHL rabbits. Migration of known standard prostaglandins coinjected with the extracts are shown above the chromatograms. The retention times of the prostaglandin standards were determined by measuring UV absorbance at 192 nm.
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Resolution of the HETEs from aortic tissues of NZW and WHHL rabbits was accomplished by normal phase HPLC using solvent system I l l . Figure 3 identifies 2 radioactive HETE peaks from aortas of 1 and 12 month old NZWs and WHHL rabbits that corresponded to the retention times of authentic 12- and 15-HETE standards. In addition, aortic tissues from 1 month old NZW and WHHLs produced a radioactive peak that migrated at a similar time as the authentic 11-HETE standard. At one month of age, the greatest amount of radioactivity detected in aortic incubations was in the 15-HETE peak. By 12 months of age, the total amount of radioactivity detected in aortic incubations was decreased with the greatest amount of radioactivity measured in the 12-HETE peak.
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80
120
160
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160
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120
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Figure 3: Metabolism of 14C-AA by aortas of 1 and 12 month old NZW and WHHL rabbits. The retention times of the known standard HETEs shown above were determined by measuring UV absorbance at 235 nm.
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GC-MS Analysis Further i d e n t i f i c a t i o n of the HETE products of WHHL and NZW rabbit aortas was accomplished by GC-MS. In Figure 4, the top panel shows the elution p r o f i l e of the radioactive products from a pool of ten aortas of 1 month old NZW rabbits. The bottom panel shows the corresponding UV absorbance at 235 nm. Similar results were observed in aortic tissue from 12 month old NZWs and 1 and 12 month old WHHLs (data not shown). The radioact i v e peaks numbered 1-6 were collected and GC-MS of the methyl ester-TMS ether derivatives performed.
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Figure 4: I d e n t i f i c a t i o n of HETEs synthesized by aortas o? one month old NZW rabbits. Following incubation of aortas obtained from ten i month old NZW rabbits, buffer was extracted and products separated by RP-HPLC using solvent system I. Fractions 100-130 were pooled, extracted with cyclohexane/ethyl acetate and rechromatographed on normal phase HPLC using solvent system I l l . The retention times of the known standard HETEs are shown above the chromatogram.
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The GC-MS results are shown in Table 1. Peaks 1-6 had UV absorption maxima at 235 nm indicating the presence of a conjugated diene. In addition, the methyl ester-TMS ether derivatives of peaks I-6 eluted from the GC column at 7.5 min which corresponded to the elution times of the methyl ester-TMS ether derivatives of authentic 12-, 15-, 11-, 9-, 8- and 5-HETE standards. The mass-spectra of the methyl ester-TMS ether derivatives of peaks 1-6 revealed the presence of a mass ion at an m/e of 406 as well as major ion fragments at 391 (loss of -CH3) and 316 (loss of Me3SiOH). Peak i was identified as 12-HETE on the basis of the presence of a major fragment ion at 295 [MCH2-CH=CH(CH2)4-CH3]. The identification of peak 2 as 15-HETE was based on the presence of characteristic ions at 335 [M- (CH2)4-CH3] and 225 [MCH2-CH=CHCH2-CH=CH-(CH2)3]. In addition, peaks 3-6 were identified by fragmentation patterns that corresponded to 11-, 9-, 8-, and 5-HETE, respectively.
TABLE I
MAJOR FRAGMENT IONS OF HETE METHYL ESTER-TMS ETHER DERIVATIVES OBTAINED FROMHPLC FRACTIONS (FIGURE 4) OF AORTAS FROM 1 MONTHOLD NZW RABBITS
Peak No.
UV Absorbance HETE maxima (nm)
M/E of Major Fragment Ions
I
235
12
295(100),316(9),391(2),406(0.6)
2
235
15
225(i00),316(24),335(17),391(2),406(0.4)
3
235
11
225(100),316(9),391(i),406(0.4)
4
235
9
255(100),316(4),391(0.6),406(0.1)
5
235
8
265(100),316(3),391(0.6),406(0.1)
6
235
5
129(100),203(15),255(168),305(24),316(12), 391(3),406(0.8)
Values in parenthesis represent the relative abundances of ions.
characteristic
Although the data are not shown, similar fragmentation patterns were observed when peaks 1-6 were analyzed from aortas of 12 month NZW and 1 and 12 month WHHL rabbits. In summary, aortas from 1 and 12 month old NZW and WHHL rabbits synthesized 12-, 15-, 11-, 9-, 8-, and 5-HETE based on comigration of the radioactive peaks with known standards, UV absorbance at 235 nm and typical mass spectra for the methyl ester-TMS derivatives (Figure 4, Table 1). It should be noted that 12-, 15- and 11-HETE were the major HETE products from
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aortic tissues of 1 month old rabbits. Aortas from 12 month old rabbits produced 12- and 15-HETE as their major HETE products. The other HETEs were minor metabolites that were only detected when aortic tissues from ten animals were pooled and analyzed. Effects of Inhibitors on Arachidonic Acid Metabolism Pretreatment of aortic tissue with the cyclooxygenase i n h i b i t o r , indomethacin (10"5M) abolished PG formation but increased the synthesis of 12- and 15-HETE (Figure 5). Removal of the endothelium from the aortas of 1 month old NZW rabbits inhibited the synthesis of both PGs and HETEs (Figure 5). Addit i o n a l l y , the production of PGs and HETEs was abolished by pretreatment of aortas with either ETYA or NDGA (Figure 6). Similar results with indomethacin, ETYA, NDGA and removal of the endothelium were observed in aortic tissues from 12 month old NZW and 1 and 12 month old WHHL rabbits (data not shown).
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HETES w uJ
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Figure 5: Metabolism of 14C-AA by aortas of 1 month old NZW rabbits. Following incubation, buffer was extracted, PGs were resolved by RP-HPLC using solvent system II ( l e f t panel) and HETEs were resolved by normal phase HPLC using solvent system I l l (right panel). Migration of known standard eicosanoids are shown above.
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OCTOBER 1988 VOL. 36 NO. 4
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o
HETES
PGS
c
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L
t55
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Figure 6: Metabolism of 14C-AA by aortas of 1 month old NZW rabbits. Following incubation, buffer was extracted, PGs were resolved by RP-HPLC using solvent system II ( l e f t panel) and HETEs were resolved by normal phase HPLC using solvent system I I I (right panel). Migration of known standard eicosanoids are shown above. Q u a n t i t a t i o n of 6-Keto PGFlo , 12-HETE and 15-HETE Production
Although the HPLC results obtained from incubating aortas from NZW and WHHL rabbits suggested that differences in aortic AA metabolism may exist between WHHL and NZW rabbits, i t is very d i f f i c u l t to quantitate the results from HPLC data. Therefore, the next series of experiments were designed to measure more accurately the production of 6-keto PGFIa, 12-HETE and 15-HETE. Table 2 shows the synthesis of 6-keto PGFI~ by aortas of i and 12 month old NZW and WHHL rabbits incubated with AA (IO-~M), A23187 (20pM) or buffer alone.
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TABLE 2 SYNTHESIS OF 6-KETO PGFIM BY AORTASOF 1 AND 12 MONTH OLD NZW AN~ WHHLRABBITS Control Group
Arachidonic Acid (pg/mg)
NZW i month 12 month
293 ± 27 426 ± 28a
1201 ± 46* 1229 ± 71"
WHHL i month 12 month
266 ± 25 265 ± 27
1327 ± 68* 545 ± 43*b
A23187
1691 ± 10~*c 1265 ± 751262 ± 145" 439 ± 29*b
Segments of thoracic aortas were incubated for 30 minutes with arachidonic acid (10-4M) or A23187 (20 ~M). 6-Keto PGFla was measured in the buffer by RIA. Data are expressed as mean ± SEM for n = 10. "'p < 0.001 control vs treatment; a = p < 0.01 12 month NZW control vs 12 month WHHL control and 1 month NZW control; b : p < 0.001 12 month WHHL treatment vs 12 month NZW treatment and 1 month WHHL treatment, c= p < 0.05 I month NZW A23187 t r e a t ment vs 12 month NZW A23187 treatment and 1 month WHHL A23187 treatment. Incubation of aortas from 1 and 12 month old NZW and WHHL rabbits with e i t h e r AA or A23187 caused an increase in 6-keto PGFla production; however, the stimulation was less in 12 month old rabbits. In vessels obtained from 1 month old WHHL and NZW rabbits, AA or A23187 e l i c i t e d an approximate 5-fold stimulation, of 6-keto. PGF.l a . Pr°ducti°n" The production of 6-keto PGFla was increased 3 - f o l d in a o r t l c tlssue from 12 month old NZW rabbits incubated with e i t h e r AA or A23187. In comparison, atherosclerotic aortas from 12 month old WHHL rabbits e l i c i t e d only a 2 - f o l d increase in 6-keto PGFla production in response to AA or A23187. Moreover, when stimulating the tissue with e i t h e r AA or A23187, the absolute value f o r a o r t i c 6-keto PGFI~ in 12 month old WHHL rabbits was approximately 60% less than that measured ~n a o r t i c tissue from a l l other groups of rabbits. I t is also important to note that the control synthesis of 6-keto PGFIa was reduced in 12 month old WHHL rabbits in comparison to 12 month old NZW rabbits (Table 2). The production of 12-HETE and 15-HETE measured in HEPES buffer obtained from a o r t i c tissue incubated under control conditions ( i . e . , no stimulation with AA or A23187) is shown in Table 3.
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TABLE 3 SYNTHESIS OF 12-HETE AND 15-HETE BY AORTASOF 1 AND 12 MONTH OLD NZW AND WHHLPJ~BBITS 12-HETE Group
15-HETE (pg/mg)
NZW I month 12 month
4 ± 1 12 ± 2b
WHHL 1 month 12 month
5 ± 1 9 ± 2b
110 ± 12 nd a 34 ± 5* 4 ± 0.3a
Segments of thoracic aortas were incubated in HEPES buffer for 30 minutes at 25°C. 12-HETE and 15-HETE were measured in the buffer by specific RIAs. Data are expressed as mean ± SEM for n = 8. n d = not detectable, p < 0.001 1 month NZW vs i month WHHL for 15-HETE. a = p < 0.001 12 month NZW and WHHL vs i month NZW and WHHL for 15-HETE. b = p < 0.01 12 month NZW and WHHL vs 1 month NZW and WHHL for 12-HETE. Aortas from i month old WHHL and NZW rabbits produced greater amounts of 15-HETE than 12-HETE. Although 12-HETE production was similar in aortas obtained from i month old NZW and WHHL rabbits, aortas from 1 month old WHHL rabbits produced approximately 70% less 15-HETE than did aortas obtained from i month old NZW rabbits. As the rabbits aged, the production of 15-HETE was depressed in aortas obtained from both WHHL and NZW rabbits. In contrast, aortic 12-HETE production in 12 month old rabbits increased s l i g h t l y in comparison to that measured in aortas from i month old rabbits. No s i g n i f icant differences in either 12-HETE or 15-HETE production were measured between aortas obtained from 12 month old WHHL and NZW rabbits. DISCUSSION The present experiments demonstrate that AA was metabolized via cyclooxygenase enzymes to PGs in aortic tissues of both WHHL and NZW rabbits. The major metabolites were 6-keto PGFla and PGE2. As WHHL rabbits developed atherosclerotic lesions, there was a reduced a b i l i t y of aortas to synthesize 6-keto PGFIa as measured by RIA. These results are in agreement with data from other studies in cholesterol-fed rabbits in which the development of atherosclerosis was accompanied by a suppression in aortic 6-keto PGFla production ( i - 4 ) . Because the vascular endothelium is the major source of PGI.2 in blood vessels (23), functional alterations in the endothelium, precipItated by the occurrence of atherosclerosis in aortas of WHHL rabbits, may explain the decreased synthesis of 6-keto PGFIa. Aortas from WHHL rabbits have a reduced capacity to produce endothelium-derived relaxing factor in response to acetylcholine (24) suggesting that there is some dysfunction of the vascular endothelium. In addition to the endothelial cells of vessels,
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vascular smooth muscle cells in culture have been shown to synthesize PGI2 (23). Larrue et al. (25) demonstrated that aortic smooth muscle cells cultured from cholesterol-fed atherosclerotic rabbits exhibited a reduced formation of 6-keto PGF ~ in comparison to aortic smooth muscle cells cultured from normal rabblts. ~ a recent study by Ecsedi and Virag (26), smooth muscle cells from aortas of WHHL rabbits were grown in culture and shown to d i f f e r morphologically from smooth muscle cells of normal rabbit aorta. Thus, i t is probable that alterations in both the endothelium and smooth muscle cells contribute to the reduced synthesis of 6-keto PGFla in aortas from WHHL rabbits. Finally, the decrease in PG production may also reflect a depressed a c t i v i t y of the cyclooxygenase enzyme in aortic tissue of 12 month old WHHL rabbits. Although aging increased the basal production of 6-keto PGFIa in aortas of NZW rabbits, the a b i l i t y of aortas to synthesize 6-keto PGFla in response to stimulation with either AA or A23187 was reduced in comparison to the enhanced stimulation of 6-keto PGFla production by aortas of 1 month old NZW rabbits. Other studies have also reported a decreased vascular production of prostaglandins in older animals (27-28). The mechanisms whereby the reduction in 6-keto PGFla production occurs in aortas from old NZW rabbits may be similar to those suggested for the observed decreased production of 6-keto PGFI~ in atherosclerotic WHHL rabbit aortas, i . e . changes in vascular endothellum, vascular smooth muscle or cyclooxygenase a c t i v i t y . Findings from this study demonstrate the a b i l i t y of aortas from both age groups of WHHL and NZW rabbits to synthesize 12-, 15-, 11-, 9-, 8- and 5-HETE. 12-HETE and 15-HETE were the major metabolites. An earlier study had shown that normal rabbit aortas synthesized only 12-HETE (16) while a later study by Henrikkson et al ( 2 9 ) reported that 15-HETE was synthesized by aortas of cholesterolfed rabbits and not by aortas of normal rabbits. Funk and Powell (18) demonstrated that rabbit aortas synthesized only 12-, 15- and 11-HETE. A possible reason for the differences in results from these studies may relate to the quantity of aortic tissue used for HETE characterization as well as to the age of the animals studied. In the present experiments, i t was necessary to pool aortas from ten rabbits in each group in order to obtain enough material for complete and accurate characterization of the HETEs by GC-MS. When lesser amounts of tissue were incubated with AA and A23187, only 12- and 15-HETE could be detected in the aortas of 12 month old WHHL and NZW rabbits while 12-, 15- and 11-HETE were produced by aortas of i month old rabbits. Pretreatment with indomethacin increased total HETE synthesis whereas ETYA and NDGA treatment blocked HETE production indicating that the HETEs are lipoxygenase metabolites of AA. When measured by RIA, 15-HETE was the major HETE synthesized by 1 month old animals but by 12 months of age, 15-HETE synthesis was suppressed such that 12-HETE was then the major product. 15-HETE is the major HETE synthesized by cultured endothelial and vascular smooth muscle cells (30). Alterations in the endothelium and/or smooth muscle of aortas from 12 month old rabbits may explain the depression in aortic 15-HETE production. Furthermore, the reduced synthesis of 15-HETE in aortas obtained from i month old WHHL rabbits in comparison to aortas obtained from age-matched NZW rabbits suggests that changes in the vascular endothelium and/or smooth muscle may occur in aortas of WHHL rabbits prior to the development of atherosclerosis. Alternatively, previous studies have demonstrated that 15-HETE is incorporated into the phospholipids of macrophages (31,32). I t is possible that this phenomenon may occur in macrophages/foamcells of 12 month old WHHL rabbits resulting in the apparent reduction of aortic 15-HETE production. This phenomenon may also explain the decreased synthesis of 15-HETE by aortas
528
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of 1 month old WHHL in comparison to 1 month old NZW rabbits since the presence of adhering leukocytes has been reported in young WHHL aortas (33,34). The slight increase in aortic 12-HETE synthesis in 12 month old rabbits may be attributable to the i n f i l t r a t i o n of adhering cells (such as macrophages and platelets) that produce as their major product, 12-HETE (8,35). While the existence of such cells have been demonstrated in WHHL atherosclerotic aortas, these cells do not adhere to normal aortas of older NZW rabbits (33). Therefore, additional studies are required to determine the mechanism whereby 12-HETE production increases in 12 month old rabbit aortas. An important functional consideration of the HETEs produced by aortas of WHHLs and NZWs may involve the role of these compounds in inhibiting PGI2 synthetase. HPETEs, the precursors of the HETEs, have been shown to i n h i b i t PGI2 synthetase (11,12). I f these compounds are increased in atherosclerotic aortas, i t may explain the observed decrease in PGI2 synthesis. However, the results from the present study show that HETE synthesis was depressed in aortas that also exhibited a decreased synthesis of 6-keto PGFIa suggesting that HETEs may not be responsible for blocking PGI2 production. Likewise, in aortas from 1 month old WHHL rabbits that exhibiteda reduced synthesis of 15HETE, 6-keto PGFla production was unchanged in comparison to production by aortas of i month old NZW rabbits. In summary, the present work represents the f i r s t reported study to characterize AA metabolism in aortas obtained from various age groups of WHHL and NZW rabbits. The major PG produced by WHHL and NZW aortas was 6-keto PGFIa, with lesser amounts of PGE2. WHHLand NZW aortas produced primarily 12- and 15-HETE. As animals aged and as atherosclerosis progressed, aortas lost the a b i l i t y to synthesize 6-keto PGFla and 15-HETE. Prior to any visible signs of aortic atherosclerosis, vesseTs obtained from i month old WHHL rabbits produced 70% less 15-HETE than did aortas obtained from age-matched control NZW rabbits. In addition, atherosclerotic aortas obtained from 12 month old WHHL rabbits synthesized lesser amounts of 6-keto PGFIa than did non-atherosclerotic aortas of age-matched control NZW rabbits. Although the mechanism by which the reduction of synthesis occurs is not known at this time, the development and expression of atherosclerosis in WHHL rabbits appears to impair the a b i l i t y of aortas to metabolize AA to both PGs and HETEs. BecauseHETE synthesis is depressed in aortas that exhibit a decreased synthesis of 6-keto PGFlq, this study also suggests that vascular HETEs are not responsible for inhiblting PGI2 production in atherosclerotic aortas. ACKNOWLEDGEMENTS The authors with to thank Mrs. Martha Williams and Mr. Milton Brady for their technical assistance and Ms. Debbie Shuttlesworth for her secretarial assistance. Support was provided by a grant from the National, Heart, Lung and Blood Institute (HL-31581) and the Moss Heart Fund, Dallas, TX. The Finnigan Mass Spectrometer was purchased with funds provided by the National Institutes of Health (GM-27506 and GM-16488-51). Dr. Campbell is the recipient of a Research Career Development Award from the National Institutes of Health (KO4-HL-O0801).
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REFERENCES
1.
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2.
Dembinska-Kiec, A., Gryglewski, T., Zmuda, A. and Gryglewski, R.J. The generation of prostacyclin by arteries and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits. Prostaglandins 14:1025, 1977.
3.
Gryglewski, R.J., Dembinska-Kiec, A., Zmuda, A. and Gryglewski, J. Prostacyclin and thromboxane A? biosynthesis capacities of heart, arteries and platelets at various ~tages of experimental atherosclerosis in rabbits. Atherosclerosis 31:385, 1978.
4.
Tremoli, E., Socini, A., Petroni, A. and Galli, C. Increased platelet aggregability is associated with increased prostacyclin production by vessel walls in hypercholesterolemic rabbits. Prostaglandins 2_44:397, 1982.
5.
D'Angelo, V., V i l l a , S., Mysliwiec, M., Donati, M.B. and deGaetano, G. Defective f i b r i n o l y t i c and prostacyclin-like a c t i v i t y in human atheromatous plaques. Thromb Haemostas 39:535, 1978.
6.
Sinzinger, H., Winter, M., Feigel, W., Silberbauer, K., and Averswald, W. Prostacyclin (PGI2)-generation by different types of human atherosclerotic lesions. Lancet II:469, 1979.
7.
Moncada, S., Gryglewski, Rod., Bunting, S. and Vane, J.R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663, 1976.
8.
Hamburg, M. and Samuelsson, B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci, USA 71:3400, 1974.
9.
Borgeat, P. and Samuelsson, B.. Arachidonic acid metabolism in polymorphonuclear leukocytes: effects of ionophore A23187. Proc Natl Acad Sci, USA 76:2148, 1979.
10.
Trachte, G.J., Lefer, A.M., Aharony, D. and Smith, J.B. Potent cons t r i c t i o n of cat coronary arteries by hydroperoxides of arachidonic acid and i t s blockade by anti-inflammatory agents. Prostaglandins 18:909, 1978.
11.
Moncada, S., Gryglewski, R.J., Bunting, S. and Vane, J.R. A l i p i d peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation. Prostaglandins 12:715, 1976.
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12.
Turk, J., Wyche, A. and Needleman, P. Inactivation of vascular prostacyclin synthetase by platelet lipoxygenase products. BiochemBiophys Res Commun 95:1628, 1980.
13.
Nakao, J., Doyama, T., Ito, H., Chang, W. and Murota, S. Comparative effect of lipoxygenase products of arachidonic acid on rat aortic smooth muscle cell migration. Atherosclerosis 44:339, 1982.
14.
Aharony, D., Smith, J.B. and Silver, M.J.. Regulation of arachidonateinduced platelet aggregation by the lipoxygenase product, 12-hydroxyperoxyeicosatetraenoic acid. Biochim Biophys Acta 718:192, 1982.
15.
Vericel, E. and Lagarde, M. 15-Hydroperoxyeicosatetraenoic acid inhibits human platelet aggregation. Lipids 1__5:472, 1980.
16.
Greenwald, J.E., Bianchine, J.R. and Wong, L . K . The production of the arachidonate metabolite HETE in vascular tissue. Nature 281:588, 1979.
17.
Herman, A.G., Claeys, M., Moncada, S. and Vane, J . R . Biosynthesis of prostacyclin (PGI2) and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) by pericardium, pleura, peritoneum, and aorta of the rabbit. Prostaglandins 1___8:439, 1979.
18.
Funk, C.D. and Powell, W.S. Release of prostaglandins and monohydroxy and trihydroxy metabolites of l i n o l e i c and arachidonic acid by adult and fetal aortae and ductus arteriosis. J Biol Chem 260:7481, 1985.
19.
Watanabe, Y. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL-rabbit). Incidence and development of atherosclerosis and xanthoma. Atherosclerosis 36:261, 1980.
20.
8uja, M.L., Kita, T., Goldstein, J.L., Watanabe, Y. and Brown, M.S. Cellular pathology of progressive atherosclerosis in WHHL rabbit. An animal model of familial hypercholesterolemia. Arteriosclerosis 3:87, 1983.
21.
Powell, W.S. Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl s i l i c a . Prostaglandins 20:947, 1980.
22.
Campbell, W.B. and Ojeda, S.R. Measurement of prostaglandins by radioimmunoassay. Methods in Enzymology14__1:323, 1987.
23.
Baenziger, N.L., Bechever, P.R. and Majerus, P.W. Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts. Cell 166:967, 1979.
24.
Wines, P.A., Schmitz, J.M., Willerson, J.T. and Campbell, W.B.. Augmented vasoconstrictor responses to serotonin precede the development of atherosclerosis in the aorta of the WHHL rabbit. Manuscript submitted for publication.
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25.
Larrue, J., Leroux, C., Darer, D. and Bricaud, H. Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta. Biochim 8iophys Acta 710:257, 1982.
26.
Ecsedi, G.G. and Virag, S. Accumulation of cholesterol ester in cultured smooth muscle cells of congenital hypercholesterolemic (WHHL) rabbits treated with normal and WHHLrabbit plasma LDL. Meth and Find Exptl Clin Pharmacol. 8:535, 1986.
27.
Kent, R.S., Kitchell, B.B., Shand, D.G. and Whorton, A.R. The a b i l i t y of vascular tissue to produce prostacyclin decreases with age. Prostaglandins 21:483, 1981.
28.
Chang, W.C. and Tai, H.H. Changes in arachidonate metabolism in aortas and platelets in aging rats. Prostaglandins Leuko Med 12:149, 1983.
29.
Henricksson, P., Hamberg, M. and Diczfalusy, U. Formationof 15-HETE as a major hydroxyeicosatetraenoic acid in atherosclerotic vessel wall. Biochim Biophys Acta 834:272, 1985.
30.
Larrue, Jo, Bricaud, H. donic acid Biophys Res
31.
Pawlowski, N.A., Scott, W.A., Andreach, M. and Cohn, Z . A . Uptake and metabolism of monohydroxyeicosatetraenoic acid by macrophages. J Exp Med 155:1653, 1982.
32.
Stenson, W.F., Nickells, M.W. and Atkinson, J.P. Esterification of monohydroxy fatty acids into the lipids of a macrophage cell line. Prostaglandins 26:253, 1983.
33.
Clubb, F.J., Stegner, J.G., Fields, H.A., Willerson, J.T. and Buja, L.M. Vascular changes in the young Watanabe Heritable Hyperlipidemic (WHHL) rabbit. Fed Proc (ABSTRACT)45:812, 1986.
34.
Butler, M.M., Clubb, F.J., Stegner, J.G., Willerson, J.T. and Buja, L.M. Surface distribution of leukocytes and foam cells in young Watanabe Heritable Hyperlipidemic (WHHL) rabbit aortas. Fed. Proc. {Abstract) 46:977, 1987.
35.
Mathur, S.N., Field, F.J., Spector, A.A. and Armstrong, M.L. Increased production of lipoxygenase products by cholesterol-rich macrophages. Biochim Biophys Acta 83__Z7:13, 1985.
Rigaud, M., Razaka, G., Daret, D., Demond-Henri, J. and Formation of monohydroxyeicosatetraenoic acids from arachiby cultured rabbit aortic smooth muscle cells. Biochem Commun 11___22:242, 1983.
Editor: G. Kaley
532
Received: 11-23-87
Accepted: 8-18-88
OCTOBER 1988 VOL. 36 NO. 4
WHHL rabbits develop progressive atherosclerosis. There are no visible signs of the disease at 1 month, however, by 12 months, the formation of aortic plaques is extensive. This study characterized arachidonic acid (AA) metabolism in 1 and 12 month old WHHL and NZW rabbit aortas. Vessels incubated with 14C-AA and A23187 metabolized AA to a number of oxygenated products as identified by high pressure liquid chromatography. The major AA metabolites produced by WHHL and NZW aortas were 6-keto PGFI~, PGE2, 12- and 15-hydroxyeicosatetraenoic acids (HETEs). The structures o~ the HETEs were confirmed by gas chromatography-mass spectrometry. Indomethacin blocked the synthesis of prostaglandins (PGs) but not HETEs whereas ETYA, NDGAor removal of the endothelium attenuated the production of b o t h PGs and HETEs. Measurement of 6-keto PGF~a, 12- and 15-HETE by specific radioimmunoassays indicated that as the rabblts aged and as atherosclerosis progressed, aortas lost the a b i l i t y to synthesize 6-keto PGFla and 15-HETE. Prior to the development of atherosclerosis, 1 month old WHHL aortas produced 70% less 15-HETE than did NZW aortas. Atherosclerotic aortas from 12 month old WHHLs synthesized 60% less 6-keto PGFla during stimulation with AA or A23187 than did 12 month old NZW aortas. We conclude that the development and expression of atherosclerosis in WHHL rabbits impairs the a b i l i t y of aortas to metabolize AA to both PGs and HETEs. INTRODUCTION
Studies have suggested that AA metabolism is altered in vessels obtained from atherosclerotic rabbits (1-4). In rabbits fed a high cholesterol diet to induce atherosclerosis, aortas as well as coronary and mesenteric arteries have a decreased a b i l i t y to synthesize prostacyclin (PGI2) (1-2,4). Platelets obtained from these animals produce increased amounts of thromboxane A2 (TxA~) (3). Based on these results as well as similar results in other specles, including man (5,6), i t is hypothesized that an imbalance between PGI2, a vasodilator and inhibitor of platelet aggregation, and TxA2, a vasoconstrictor and promoter of platelet aggregation, contributes to the expression of atherosclerosis (3,7).
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In addition to the cyclooxygenase pathway, AA is metabolized by lipoxygenase enzymes to form hydroperoxyeicosatetraenoicacids (HPETEs) which are then reduced to the HETEs (8,9). HPETEs (and to a lesser extent, HETEs) have a v a r i e t y of biological actions, including vasoconstriction (10), i n h i b i t i o n of PGI2 synthetase (11,12), migration of aortic smooth muscle cells (13), and i n h i b i t i o n of p l a t e l e t aggregation (14,15). Previous studies have demonstrated that vessels from normal rabbits have the capacity to synthesize HETEs (16-18); however, the production of these compounds has not been well characterized. Likewise, the a b i l i t y of vessels from atherosclerotic rabbits to metabolize AA to HETEs has not been studied. The production of HPETEs and HETEs may be altered in atherosclerosis, and may, for example, be responsible for the decreased production of PGI2 in atherosclerotic rabbit vessels. The WHHL rabbit is an inbred strain of rabbits characterized by a deficiency in functional cell surface receptors for low density lipoproteins. The rabbits develop hypercholesterolemia and severe atherosclerosis (19). No v i s i b l e signs of the disease are seen at one month of age despite elevated serum cholesterol concentrations. By 3 months of age, there is gross evidence of atherosclerosis, and at 12 months of age, the formation of atheromatous plaques and foam cell lesions in the aorta is extensive (20). Import a n t l y , the characteristics of atherosclerosis in the WHHL rabbit more closely resembles human f a m i l i a l hypercholesterolemia than does the d i e t induced atherosclerosis observed in cholesterol-fed rabbits (20). WHHL rabbits are therefore a good model in which to investigate the metabolism of AA in vascular tissue and to determine i f the development of atherosclerosis in WHHL rabbits is accompanied by alterations in AA metabolism. The present study characterizes the production of PGs and HETEs in aortas obtained from i and 12 month old WHHL rabbits and compares this to the production of PGs and HETEs in aortic tissue of age-matched control NZW rabbits.
METHODSAND MATERIALS Experimental Animals Male and female NZW rabbits ( i and 12 months of age) were purchased from Hickory H i l l . Male and female homozygous WHHL rabbits (1 and 12 months of age) were obtained from our colony at Dallas. B o t h groups of animals ate standard laboratory rabbit chow. Rabbits were sacrificed by cervical dislocation, and the thoracic aorta was rapidly removed. The vessels were carefully cleaned of a l l adhering f a t and connective tissue and placed in HEPES buffer (0.01 M HEPES, 0.15 M NaCl, 0.005 M KCI, 0.002 M CaCl2, 0.001 M MgCl2, 0.006 M glucose, pH 7.4). The aortas were cut open l o n g i t u d i n a l l y and then sliced with a razor blade into approximate 2-3 mm. wide segments taking care not to disrupt the endothelium. Incubation of Aortic Tissue with Arachidonic Acid
Strips of vessels from each age group of WHHL and NZW rabbits (100 mg of wet weight) were incubated in 1 ml of HEPES buffer (pH 7.4) at 37°C for 15 minutes with 14C-AA (0.05 NCi, 10-7M) and the calcium ionophore, A23187 (20 NM). Parallel incubations were performed under the same conditions but in the absence of aortic tissue to assess the extent, i f any, of auto-oxidation of AA. In a separate series of experiments, vessels from 1 and 12 month old
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NZW and WHHL rabbits were preincubated for 10 minutes with various i n h i b i t o r s (indomethacin, [I0-5M], 5,8,11,14-eicosatetraynoic acid (ETYA),[3 x 10-5M]~ nordihydroguaiaretic acid (NDGA), [5 x 10-~]) or the endothelium was removeo by gentle ru)bing of the intimal surface with a cotton swab, p r i o r to incubation with 14C-AA and A23187. In another set of experiments designed to i d e n t i f y AA metabolites by gas chromatography-mass spectrometry, aortic tissue from ten NZW and WHHL rabbits (at i and 12 months of age) were incubated for 15 minutes at 37°C in 100 ml of HEPES buffer containing 14C-AA (0.05 ~Ci, 5 x 10-5 ) and A23187.
Isolation and Identification of Arachidonic Acid Metabolites Following incubation, the buffer was removed from the vessels and a c i d i f i e d to pH 3.0 with glacial acetic acid. The AA metabolites were extracted over a Baker C118 octadecylsilica (ODS) column as previously described by Powell (21). The ODS column was washed sequentially with 5 ml ethanol and water. The a c i d i f i e d sample (made 15% [ v o l / v o l ] with respect to ETOH) was then added to the column, and the column washed with 5 ml of 15% ethanol, followed by 5 ml water and f i n a l l y by 5 ml petroleum ether. In some experiments, the HETE fraction was eluted with 10 ml chloroform/petroleum ether (50:50) followed by elution of the PGs with 6 ml ethyl acetate. In other experiments, the HETEs and PGs were eluted together with 6 ml ethyl acetate. Recovery of the PGs and HETEs by this extraction procedure was greater than 95%. The extracted samples were evaporated under a nitrogen stream and stored at -40°C until analysis by high pressure l i q u i d chromatography (HPLC). For reverse-phase (RP) HPLC, a C18 column, (RP-18, 5N, 4.6 x 250 mm, Unimetrics) was used. Two d i f f e r e n t solvent systems were employed. In solvent system 1, solvent A was water and solvent B was a c e t o n i t r i l e containing 0.1% glacial acetic acid. The program was a 40 minute linear gradient at a flow rate of i ml/min from 50% solvent B in A to 100% solvent B. The sample was redissolved and injected in 200 Nl of a c e t o n i t r i l e : w a t e r : acetic acid (50:50:0.01). The eluate was collected in 0.2 ml aliquots and r a d i o a c t i v i t y measured. In solvent system I I , solvent A was water containing 0.025 M phosphoric acid and solvent B was a c e t o n i t r i l e . The program consisted of a 40 minute isocratic phase with 31% B in A, followed by a 20 minute linear gradient to 100% B and 20 minute isocratic phase with 100% B. Flow rate was 1 ml/min. The sample was redissolved and injected in 200 Nl of acetonitrile:water:phosphoric and (31:69:0.01). The eluate was collected in 0.5 ml aliquots and r a d i o a c t i v i t y measured. For normal phase HPLC, a s i l i c a gel column (Ultrasphere, 5N, 4.6 x 250 mm, Beckman) was used. Solvent system I l l consisted of solvent A which was hexane containing 0.1% glacial acetic acid and solvent B was hexane containing 0.1% glacial acetic acid and 2% isopropanol. The program consisted of a 40 minute linear gradient from 25% solvent B in solvent A to 75% solvent B in A. The flow rate was 3 ml/min. The sample was redissolved and injected in hexane:isopropanol:acetic acid (995:5:1). The eluate was collected in 0.6 ml aliquots and r a d i o a c t i v i t y measured.
Gas ChromatographyMass Spectrometry (GC-MS) In order to i d e n t i f y the HETEs from aortic tissue by GC-MS, the extracted metabolites were f i r s t chromatographed on RP-HPLC using solvent system I. The fractions corresponding to the HETEs (#100-130) were c o l l e c t i v e l y pooled, acidified, extracted with cyclohexane/ethyl acetate (50:50) and
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rechromatographed on normal phase HPLC using solvent system I I I . The radioactive peaks corresponding to the retention times of the known HETE standards were collected and derivatized. First, the samples were treated with etheral diazomethane for 30 minutes on ice to form the methyl ester. The hydroxyl groups were then converted to the t r i m e t h y l s i l y l (TMS) ethers by addition of 15 Nl bis-(TMS)-trifluoroacetamide. Sampleswere incubated overnight at 40°C and solvent then removed under argon. GC-MSanalysis was performed with a Finnigan 4021 quadrupole mass spectrometer and a Finnigan INCOS 2000 data system. Ionization of the samples was done by electron impact at 65-70 eV. The column used was a 3% SP-2100-DOH on Supelcoport (6 f t , glass). The flow rate of the carrier gas (N2) was 20-30 ml/min and oven temperature was 220°C.
Radioimmunoassays (RIAs) for PGs and HETES Segments of thoracic aorta (10 mg of wet weight) were obtained from 1 and 12 month old NZW and WHHL rabbits. The vessels were incubated for 30 minutes at 25% in 1 ml of HEPES buffer containing either AA (I0-4M), A23187 (20 NM) or buffer alone. The aortic synthesis of PGI2 was measured in the buffer by RIA of i t s stable metabolite,. 6-keto PGFIa The aortic synthesis of 12-HETE and 15-HETE were measured in the buffer "by specific RIAs. The antibodies for 6-keto PGFIa and 15-HETE were produced in our laboratory in rabbits and the antibody for 12-HETE was supplied by Dr. Levine (Brandeis University). The RIA method used to measure the compounds was similar to that previously described (22). The sensitivity of the assays is less than 5 pg for 6-keto PGFIa, 12-HETE and 15-HETE. Cross-reactivity of the various antisera with known AA metabolites is less than 0.1%. Materials [14C (uniformly labeled)]-AA; (3H)-I2(S)-HETE, (3H)-I5(S)-HETE were obtained from New England Nuclear; HPLC grade solvents were from Baker; (3H)-6-keto PGFla was from Amersham; 12(S)-HETE, 15(S)-HETE and 5(S)-HETE were from BioMol; HEPES, A23187, AA, NDGA and indomethacin were from Sigma; l i q u i d s c i n t i l l a t i o n counters were from Beckman and Packard Instruments; budget solve s c i n t i l l a t i o n f l u i d was from Research Products, Inc.; 6-keto PGFI~, PGE2, TxBo, PGF~a, PGD~ were from Upjohn Co." ETYA was a g i f t from HoffmanLaRoche; bls(TMS~ trifluoracetamide was from Supelco, Inc. Statistical Analysis The RIA data were expressed as means ± SEM. Statistical evaluation of the data was performed by using Student's "t" test. RESULTS Arachidonic Acid Metabolism by Aortas of WHHL and NZW Rabbits Gross examination of aortas from I and 12 month old NZW and 1 month old WHHL rabbits appeared normal. Aortas from 12 month old WHHL rabbits showed visible f a t t y streaks and raised plaque areas. Incubation of aortas from various age groups of WHHL and NZW rabbits with 14C-AA and A23187 resulted in the metabolism of AA to a number of oxygenated products as identified by RP-HPLC using solvent system I (Figure i ) . Aortas from 1 month old WHHL and NZW rabbits produced radioactive peaks which corresponded to the elution
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PROSTAGLANDINS
times of the PG metabolites, dihydroxyeicosatetraenoic acids (diHETEs) or dihydroxyeicosatrienoic acids (DHETs), HETEs and AA. As the rabbits aged, the aortas appeared to lose the a b i l i t y to metabolize AA. As shown in Figure i , decreased radioactivity was observed in the regions corresponding to the PG metabolites and HETEs, and no radioactive peaks were evident in the diHETE-DHET region. Although the pattern of metabolites was similar between WHHL and NZW rabbits, the amount of radioactivity detected in incubations of WHHL aortas was less than that detected in incubations of NZW aortas suggesting a decreased metabolism of AA in WHHL aortas. When HEPES buffer containing 14C-AA and A23187 was incubated without aortic tissue, extracted and subjected to HPLC, no AA metabolites were detected (data not shown).
NZW
~.
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FRACTION Figure 1: Metabolism of 14C-AA by aortas of 1 and 12 month old NZW and WHHL rabbits. Migration of known standard eicosanoids are shown above. These standards indicate the relative retention times of the known compounds and are not meant to identify the corresponding radioactive peaks.
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PROSTAGLANDINS
To further identify the PGs produced by aortas of WHHL and NZW rabbits, extracted samples were analyzed by RP-HPLC using solvent system I I (Figure 2). This system separates the cyclooxygenase metabolites of AA, whereas the more non-polar lipoxygenase products are unresolved. The retention time of the major cyclooxygenase radioactive peak in aortas from all rabbits corresponded exactly to the retention time of 6-keto PGFla. The smaller peak corresponded to the retention time of PGE2.
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Z
32
0
64
96
128
~60
FRACTION
:~ n
~_
WHHL
o
2500 ~ rn0nlh
iO00 500
Ol~-~-r ~ ~ 0 52
'
_
/~
+
J
12month
64
96
~tJ k_~r.~.., j i28 160
FRACTION
Figure 2: Metabolism of 14C-AA by aortas of 1 and 12 month old NZW and WHHL rabbits. Migration of known standard prostaglandins coinjected with the extracts are shown above the chromatograms. The retention times of the prostaglandin standards were determined by measuring UV absorbance at 192 nm.
520
OCTOBER 1988 VOL. 36 NO. 4
PROSTAGLANDINS
Resolution of the HETEs from aortic tissues of NZW and WHHL rabbits was accomplished by normal phase HPLC using solvent system I l l . Figure 3 identifies 2 radioactive HETE peaks from aortas of 1 and 12 month old NZWs and WHHL rabbits that corresponded to the retention times of authentic 12- and 15-HETE standards. In addition, aortic tissues from 1 month old NZW and WHHLs produced a radioactive peak that migrated at a similar time as the authentic 11-HETE standard. At one month of age, the greatest amount of radioactivity detected in aortic incubations was in the 15-HETE peak. By 12 months of age, the total amount of radioactivity detected in aortic incubations was decreased with the greatest amount of radioactivity measured in the 12-HETE peak.
NZW
~ ~
"
1000[
12month
Z 0 WHHL_ n,,
40
laJ I000~ 0
80
120
160
200
160
200
FRACTION
500 1
0
0
0
--
5OO O'0
40
80
120
FRACTION
Figure 3: Metabolism of 14C-AA by aortas of 1 and 12 month old NZW and WHHL rabbits. The retention times of the known standard HETEs shown above were determined by measuring UV absorbance at 235 nm.
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PROSTAGLANDINS
GC-MS Analysis Further i d e n t i f i c a t i o n of the HETE products of WHHL and NZW rabbit aortas was accomplished by GC-MS. In Figure 4, the top panel shows the elution p r o f i l e of the radioactive products from a pool of ten aortas of 1 month old NZW rabbits. The bottom panel shows the corresponding UV absorbance at 235 nm. Similar results were observed in aortic tissue from 12 month old NZWs and 1 and 12 month old WHHLs (data not shown). The radioact i v e peaks numbered 1-6 were collected and GC-MS of the methyl ester-TMS ether derivatives performed.
i 500
n,750
z
o
I 0
100
50
iO0 FRACTION
'150
20
t2
D E
50
oFo
I iO
I
/ 2 MINUTES
t
I 50
P 40
Figure 4: I d e n t i f i c a t i o n of HETEs synthesized by aortas o? one month old NZW rabbits. Following incubation of aortas obtained from ten i month old NZW rabbits, buffer was extracted and products separated by RP-HPLC using solvent system I. Fractions 100-130 were pooled, extracted with cyclohexane/ethyl acetate and rechromatographed on normal phase HPLC using solvent system I l l . The retention times of the known standard HETEs are shown above the chromatogram.
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OCTOBER 1988 VOL. 36 NO. 4
PROSTAGLANDINS
The GC-MS results are shown in Table 1. Peaks 1-6 had UV absorption maxima at 235 nm indicating the presence of a conjugated diene. In addition, the methyl ester-TMS ether derivatives of peaks I-6 eluted from the GC column at 7.5 min which corresponded to the elution times of the methyl ester-TMS ether derivatives of authentic 12-, 15-, 11-, 9-, 8- and 5-HETE standards. The mass-spectra of the methyl ester-TMS ether derivatives of peaks 1-6 revealed the presence of a mass ion at an m/e of 406 as well as major ion fragments at 391 (loss of -CH3) and 316 (loss of Me3SiOH). Peak i was identified as 12-HETE on the basis of the presence of a major fragment ion at 295 [MCH2-CH=CH(CH2)4-CH3]. The identification of peak 2 as 15-HETE was based on the presence of characteristic ions at 335 [M- (CH2)4-CH3] and 225 [MCH2-CH=CHCH2-CH=CH-(CH2)3]. In addition, peaks 3-6 were identified by fragmentation patterns that corresponded to 11-, 9-, 8-, and 5-HETE, respectively.
TABLE I
MAJOR FRAGMENT IONS OF HETE METHYL ESTER-TMS ETHER DERIVATIVES OBTAINED FROMHPLC FRACTIONS (FIGURE 4) OF AORTAS FROM 1 MONTHOLD NZW RABBITS
Peak No.
UV Absorbance HETE maxima (nm)
M/E of Major Fragment Ions
I
235
12
295(100),316(9),391(2),406(0.6)
2
235
15
225(i00),316(24),335(17),391(2),406(0.4)
3
235
11
225(100),316(9),391(i),406(0.4)
4
235
9
255(100),316(4),391(0.6),406(0.1)
5
235
8
265(100),316(3),391(0.6),406(0.1)
6
235
5
129(100),203(15),255(168),305(24),316(12), 391(3),406(0.8)
Values in parenthesis represent the relative abundances of ions.
characteristic
Although the data are not shown, similar fragmentation patterns were observed when peaks 1-6 were analyzed from aortas of 12 month NZW and 1 and 12 month WHHL rabbits. In summary, aortas from 1 and 12 month old NZW and WHHL rabbits synthesized 12-, 15-, 11-, 9-, 8-, and 5-HETE based on comigration of the radioactive peaks with known standards, UV absorbance at 235 nm and typical mass spectra for the methyl ester-TMS derivatives (Figure 4, Table 1). It should be noted that 12-, 15- and 11-HETE were the major HETE products from
OCTOBER 1988 VOL. 36 NO. 4
523
PROSTAGLANDINS
aortic tissues of 1 month old rabbits. Aortas from 12 month old rabbits produced 12- and 15-HETE as their major HETE products. The other HETEs were minor metabolites that were only detected when aortic tissues from ten animals were pooled and analyzed. Effects of Inhibitors on Arachidonic Acid Metabolism Pretreatment of aortic tissue with the cyclooxygenase i n h i b i t o r , indomethacin (10"5M) abolished PG formation but increased the synthesis of 12- and 15-HETE (Figure 5). Removal of the endothelium from the aortas of 1 month old NZW rabbits inhibited the synthesis of both PGs and HETEs (Figure 5). Addit i o n a l l y , the production of PGs and HETEs was abolished by pretreatment of aortas with either ETYA or NDGA (Figure 6). Similar results with indomethacin, ETYA, NDGA and removal of the endothelium were observed in aortic tissues from 12 month old NZW and 1 and 12 month old WHHL rabbits (data not shown).
PG._.S_S
HETES w uJ
w
w
w
.... i -- I0001
~
~
.
.
.
.
.
~ I0001 01~ 0
i
i 32
i
i 64
i
-
- J 96
FRACTION
I
i t28
~
--
.
g
~
o -i " --p 160
..... I II
I I
II 'NDOMETHAcIN
MINUS ENDOTH£LIUM
0I~ ~-~---~I ~.. , 0
45
,
~ ....
90
t35
"{80
FRACTION
Figure 5: Metabolism of 14C-AA by aortas of 1 month old NZW rabbits. Following incubation, buffer was extracted, PGs were resolved by RP-HPLC using solvent system II ( l e f t panel) and HETEs were resolved by normal phase HPLC using solvent system I l l (right panel). Migration of known standard eicosanoids are shown above.
524
OCTOBER 1988 VOL. 36 NO. 4
PROSTAGLANDINS
o
HETES
PGS
c
Z500 I 500~ w
~ 2500
I
i
I
I
i
I
I
I
I
I
I
l
1
I
I
L
L
I
I
500
1250
0
I
32
64 96 FRACTION
t2B
160
L
0
45
9O FRACTION
L
t55
I
L
t80
Figure 6: Metabolism of 14C-AA by aortas of 1 month old NZW rabbits. Following incubation, buffer was extracted, PGs were resolved by RP-HPLC using solvent system II ( l e f t panel) and HETEs were resolved by normal phase HPLC using solvent system I I I (right panel). Migration of known standard eicosanoids are shown above. Q u a n t i t a t i o n of 6-Keto PGFlo , 12-HETE and 15-HETE Production
Although the HPLC results obtained from incubating aortas from NZW and WHHL rabbits suggested that differences in aortic AA metabolism may exist between WHHL and NZW rabbits, i t is very d i f f i c u l t to quantitate the results from HPLC data. Therefore, the next series of experiments were designed to measure more accurately the production of 6-keto PGFIa, 12-HETE and 15-HETE. Table 2 shows the synthesis of 6-keto PGFI~ by aortas of i and 12 month old NZW and WHHL rabbits incubated with AA (IO-~M), A23187 (20pM) or buffer alone.
OCTOBER 1988 VOL. 36 NO. 4
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PROSTAGLANDINS
TABLE 2 SYNTHESIS OF 6-KETO PGFIM BY AORTASOF 1 AND 12 MONTH OLD NZW AN~ WHHLRABBITS Control Group
Arachidonic Acid (pg/mg)
NZW i month 12 month
293 ± 27 426 ± 28a
1201 ± 46* 1229 ± 71"
WHHL i month 12 month
266 ± 25 265 ± 27
1327 ± 68* 545 ± 43*b
A23187
1691 ± 10~*c 1265 ± 751262 ± 145" 439 ± 29*b
Segments of thoracic aortas were incubated for 30 minutes with arachidonic acid (10-4M) or A23187 (20 ~M). 6-Keto PGFla was measured in the buffer by RIA. Data are expressed as mean ± SEM for n = 10. "'p < 0.001 control vs treatment; a = p < 0.01 12 month NZW control vs 12 month WHHL control and 1 month NZW control; b : p < 0.001 12 month WHHL treatment vs 12 month NZW treatment and 1 month WHHL treatment, c= p < 0.05 I month NZW A23187 t r e a t ment vs 12 month NZW A23187 treatment and 1 month WHHL A23187 treatment. Incubation of aortas from 1 and 12 month old NZW and WHHL rabbits with e i t h e r AA or A23187 caused an increase in 6-keto PGFla production; however, the stimulation was less in 12 month old rabbits. In vessels obtained from 1 month old WHHL and NZW rabbits, AA or A23187 e l i c i t e d an approximate 5-fold stimulation, of 6-keto. PGF.l a . Pr°ducti°n" The production of 6-keto PGFla was increased 3 - f o l d in a o r t l c tlssue from 12 month old NZW rabbits incubated with e i t h e r AA or A23187. In comparison, atherosclerotic aortas from 12 month old WHHL rabbits e l i c i t e d only a 2 - f o l d increase in 6-keto PGFla production in response to AA or A23187. Moreover, when stimulating the tissue with e i t h e r AA or A23187, the absolute value f o r a o r t i c 6-keto PGFI~ in 12 month old WHHL rabbits was approximately 60% less than that measured ~n a o r t i c tissue from a l l other groups of rabbits. I t is also important to note that the control synthesis of 6-keto PGFIa was reduced in 12 month old WHHL rabbits in comparison to 12 month old NZW rabbits (Table 2). The production of 12-HETE and 15-HETE measured in HEPES buffer obtained from a o r t i c tissue incubated under control conditions ( i . e . , no stimulation with AA or A23187) is shown in Table 3.
526
OCTOBER 1988 VOL. 36 NO. 4
PROSTAGLANDINS
TABLE 3 SYNTHESIS OF 12-HETE AND 15-HETE BY AORTASOF 1 AND 12 MONTH OLD NZW AND WHHLPJ~BBITS 12-HETE Group
15-HETE (pg/mg)
NZW I month 12 month
4 ± 1 12 ± 2b
WHHL 1 month 12 month
5 ± 1 9 ± 2b
110 ± 12 nd a 34 ± 5* 4 ± 0.3a
Segments of thoracic aortas were incubated in HEPES buffer for 30 minutes at 25°C. 12-HETE and 15-HETE were measured in the buffer by specific RIAs. Data are expressed as mean ± SEM for n = 8. n d = not detectable, p < 0.001 1 month NZW vs i month WHHL for 15-HETE. a = p < 0.001 12 month NZW and WHHL vs i month NZW and WHHL for 15-HETE. b = p < 0.01 12 month NZW and WHHL vs 1 month NZW and WHHL for 12-HETE. Aortas from i month old WHHL and NZW rabbits produced greater amounts of 15-HETE than 12-HETE. Although 12-HETE production was similar in aortas obtained from i month old NZW and WHHL rabbits, aortas from 1 month old WHHL rabbits produced approximately 70% less 15-HETE than did aortas obtained from i month old NZW rabbits. As the rabbits aged, the production of 15-HETE was depressed in aortas obtained from both WHHL and NZW rabbits. In contrast, aortic 12-HETE production in 12 month old rabbits increased s l i g h t l y in comparison to that measured in aortas from i month old rabbits. No s i g n i f icant differences in either 12-HETE or 15-HETE production were measured between aortas obtained from 12 month old WHHL and NZW rabbits. DISCUSSION The present experiments demonstrate that AA was metabolized via cyclooxygenase enzymes to PGs in aortic tissues of both WHHL and NZW rabbits. The major metabolites were 6-keto PGFla and PGE2. As WHHL rabbits developed atherosclerotic lesions, there was a reduced a b i l i t y of aortas to synthesize 6-keto PGFIa as measured by RIA. These results are in agreement with data from other studies in cholesterol-fed rabbits in which the development of atherosclerosis was accompanied by a suppression in aortic 6-keto PGFla production ( i - 4 ) . Because the vascular endothelium is the major source of PGI.2 in blood vessels (23), functional alterations in the endothelium, precipItated by the occurrence of atherosclerosis in aortas of WHHL rabbits, may explain the decreased synthesis of 6-keto PGFIa. Aortas from WHHL rabbits have a reduced capacity to produce endothelium-derived relaxing factor in response to acetylcholine (24) suggesting that there is some dysfunction of the vascular endothelium. In addition to the endothelial cells of vessels,
OCTOBER 1988 VOL. 36 NO. 4
527
PROSTAGLANDINS
vascular smooth muscle cells in culture have been shown to synthesize PGI2 (23). Larrue et al. (25) demonstrated that aortic smooth muscle cells cultured from cholesterol-fed atherosclerotic rabbits exhibited a reduced formation of 6-keto PGF ~ in comparison to aortic smooth muscle cells cultured from normal rabblts. ~ a recent study by Ecsedi and Virag (26), smooth muscle cells from aortas of WHHL rabbits were grown in culture and shown to d i f f e r morphologically from smooth muscle cells of normal rabbit aorta. Thus, i t is probable that alterations in both the endothelium and smooth muscle cells contribute to the reduced synthesis of 6-keto PGFla in aortas from WHHL rabbits. Finally, the decrease in PG production may also reflect a depressed a c t i v i t y of the cyclooxygenase enzyme in aortic tissue of 12 month old WHHL rabbits. Although aging increased the basal production of 6-keto PGFIa in aortas of NZW rabbits, the a b i l i t y of aortas to synthesize 6-keto PGFla in response to stimulation with either AA or A23187 was reduced in comparison to the enhanced stimulation of 6-keto PGFla production by aortas of 1 month old NZW rabbits. Other studies have also reported a decreased vascular production of prostaglandins in older animals (27-28). The mechanisms whereby the reduction in 6-keto PGFla production occurs in aortas from old NZW rabbits may be similar to those suggested for the observed decreased production of 6-keto PGFI~ in atherosclerotic WHHL rabbit aortas, i . e . changes in vascular endothellum, vascular smooth muscle or cyclooxygenase a c t i v i t y . Findings from this study demonstrate the a b i l i t y of aortas from both age groups of WHHL and NZW rabbits to synthesize 12-, 15-, 11-, 9-, 8- and 5-HETE. 12-HETE and 15-HETE were the major metabolites. An earlier study had shown that normal rabbit aortas synthesized only 12-HETE (16) while a later study by Henrikkson et al ( 2 9 ) reported that 15-HETE was synthesized by aortas of cholesterolfed rabbits and not by aortas of normal rabbits. Funk and Powell (18) demonstrated that rabbit aortas synthesized only 12-, 15- and 11-HETE. A possible reason for the differences in results from these studies may relate to the quantity of aortic tissue used for HETE characterization as well as to the age of the animals studied. In the present experiments, i t was necessary to pool aortas from ten rabbits in each group in order to obtain enough material for complete and accurate characterization of the HETEs by GC-MS. When lesser amounts of tissue were incubated with AA and A23187, only 12- and 15-HETE could be detected in the aortas of 12 month old WHHL and NZW rabbits while 12-, 15- and 11-HETE were produced by aortas of i month old rabbits. Pretreatment with indomethacin increased total HETE synthesis whereas ETYA and NDGA treatment blocked HETE production indicating that the HETEs are lipoxygenase metabolites of AA. When measured by RIA, 15-HETE was the major HETE synthesized by 1 month old animals but by 12 months of age, 15-HETE synthesis was suppressed such that 12-HETE was then the major product. 15-HETE is the major HETE synthesized by cultured endothelial and vascular smooth muscle cells (30). Alterations in the endothelium and/or smooth muscle of aortas from 12 month old rabbits may explain the depression in aortic 15-HETE production. Furthermore, the reduced synthesis of 15-HETE in aortas obtained from i month old WHHL rabbits in comparison to aortas obtained from age-matched NZW rabbits suggests that changes in the vascular endothelium and/or smooth muscle may occur in aortas of WHHL rabbits prior to the development of atherosclerosis. Alternatively, previous studies have demonstrated that 15-HETE is incorporated into the phospholipids of macrophages (31,32). I t is possible that this phenomenon may occur in macrophages/foamcells of 12 month old WHHL rabbits resulting in the apparent reduction of aortic 15-HETE production. This phenomenon may also explain the decreased synthesis of 15-HETE by aortas
528
OCTOBER 1988 VOL. 36 NO. 4
PROSTAGLANDINS
of 1 month old WHHL in comparison to 1 month old NZW rabbits since the presence of adhering leukocytes has been reported in young WHHL aortas (33,34). The slight increase in aortic 12-HETE synthesis in 12 month old rabbits may be attributable to the i n f i l t r a t i o n of adhering cells (such as macrophages and platelets) that produce as their major product, 12-HETE (8,35). While the existence of such cells have been demonstrated in WHHL atherosclerotic aortas, these cells do not adhere to normal aortas of older NZW rabbits (33). Therefore, additional studies are required to determine the mechanism whereby 12-HETE production increases in 12 month old rabbit aortas. An important functional consideration of the HETEs produced by aortas of WHHLs and NZWs may involve the role of these compounds in inhibiting PGI2 synthetase. HPETEs, the precursors of the HETEs, have been shown to i n h i b i t PGI2 synthetase (11,12). I f these compounds are increased in atherosclerotic aortas, i t may explain the observed decrease in PGI2 synthesis. However, the results from the present study show that HETE synthesis was depressed in aortas that also exhibited a decreased synthesis of 6-keto PGFIa suggesting that HETEs may not be responsible for blocking PGI2 production. Likewise, in aortas from 1 month old WHHL rabbits that exhibiteda reduced synthesis of 15HETE, 6-keto PGFla production was unchanged in comparison to production by aortas of i month old NZW rabbits. In summary, the present work represents the f i r s t reported study to characterize AA metabolism in aortas obtained from various age groups of WHHL and NZW rabbits. The major PG produced by WHHL and NZW aortas was 6-keto PGFIa, with lesser amounts of PGE2. WHHLand NZW aortas produced primarily 12- and 15-HETE. As animals aged and as atherosclerosis progressed, aortas lost the a b i l i t y to synthesize 6-keto PGFla and 15-HETE. Prior to any visible signs of aortic atherosclerosis, vesseTs obtained from i month old WHHL rabbits produced 70% less 15-HETE than did aortas obtained from age-matched control NZW rabbits. In addition, atherosclerotic aortas obtained from 12 month old WHHL rabbits synthesized lesser amounts of 6-keto PGFIa than did non-atherosclerotic aortas of age-matched control NZW rabbits. Although the mechanism by which the reduction of synthesis occurs is not known at this time, the development and expression of atherosclerosis in WHHL rabbits appears to impair the a b i l i t y of aortas to metabolize AA to both PGs and HETEs. BecauseHETE synthesis is depressed in aortas that exhibit a decreased synthesis of 6-keto PGFlq, this study also suggests that vascular HETEs are not responsible for inhiblting PGI2 production in atherosclerotic aortas. ACKNOWLEDGEMENTS The authors with to thank Mrs. Martha Williams and Mr. Milton Brady for their technical assistance and Ms. Debbie Shuttlesworth for her secretarial assistance. Support was provided by a grant from the National, Heart, Lung and Blood Institute (HL-31581) and the Moss Heart Fund, Dallas, TX. The Finnigan Mass Spectrometer was purchased with funds provided by the National Institutes of Health (GM-27506 and GM-16488-51). Dr. Campbell is the recipient of a Research Career Development Award from the National Institutes of Health (KO4-HL-O0801).
OCTOBER 1988 VOL. 36 NO. 4
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PROSTAGLANDINS
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Gryglewski, R.J., Dembinska-Kiec, A., Zmuda, A. and Gryglewski, J. Prostacyclin and thromboxane A? biosynthesis capacities of heart, arteries and platelets at various ~tages of experimental atherosclerosis in rabbits. Atherosclerosis 31:385, 1978.
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8.
Hamburg, M. and Samuelsson, B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci, USA 71:3400, 1974.
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11.
Moncada, S., Gryglewski, R.J., Bunting, S. and Vane, J.R. A l i p i d peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation. Prostaglandins 12:715, 1976.
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12.
Turk, J., Wyche, A. and Needleman, P. Inactivation of vascular prostacyclin synthetase by platelet lipoxygenase products. BiochemBiophys Res Commun 95:1628, 1980.
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Nakao, J., Doyama, T., Ito, H., Chang, W. and Murota, S. Comparative effect of lipoxygenase products of arachidonic acid on rat aortic smooth muscle cell migration. Atherosclerosis 44:339, 1982.
14.
Aharony, D., Smith, J.B. and Silver, M.J.. Regulation of arachidonateinduced platelet aggregation by the lipoxygenase product, 12-hydroxyperoxyeicosatetraenoic acid. Biochim Biophys Acta 718:192, 1982.
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Vericel, E. and Lagarde, M. 15-Hydroperoxyeicosatetraenoic acid inhibits human platelet aggregation. Lipids 1__5:472, 1980.
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Greenwald, J.E., Bianchine, J.R. and Wong, L . K . The production of the arachidonate metabolite HETE in vascular tissue. Nature 281:588, 1979.
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Herman, A.G., Claeys, M., Moncada, S. and Vane, J . R . Biosynthesis of prostacyclin (PGI2) and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) by pericardium, pleura, peritoneum, and aorta of the rabbit. Prostaglandins 1___8:439, 1979.
18.
Funk, C.D. and Powell, W.S. Release of prostaglandins and monohydroxy and trihydroxy metabolites of l i n o l e i c and arachidonic acid by adult and fetal aortae and ductus arteriosis. J Biol Chem 260:7481, 1985.
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Watanabe, Y. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL-rabbit). Incidence and development of atherosclerosis and xanthoma. Atherosclerosis 36:261, 1980.
20.
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Powell, W.S. Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl s i l i c a . Prostaglandins 20:947, 1980.
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Baenziger, N.L., Bechever, P.R. and Majerus, P.W. Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts. Cell 166:967, 1979.
24.
Wines, P.A., Schmitz, J.M., Willerson, J.T. and Campbell, W.B.. Augmented vasoconstrictor responses to serotonin precede the development of atherosclerosis in the aorta of the WHHL rabbit. Manuscript submitted for publication.
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Larrue, J., Leroux, C., Darer, D. and Bricaud, H. Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta. Biochim 8iophys Acta 710:257, 1982.
26.
Ecsedi, G.G. and Virag, S. Accumulation of cholesterol ester in cultured smooth muscle cells of congenital hypercholesterolemic (WHHL) rabbits treated with normal and WHHLrabbit plasma LDL. Meth and Find Exptl Clin Pharmacol. 8:535, 1986.
27.
Kent, R.S., Kitchell, B.B., Shand, D.G. and Whorton, A.R. The a b i l i t y of vascular tissue to produce prostacyclin decreases with age. Prostaglandins 21:483, 1981.
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Chang, W.C. and Tai, H.H. Changes in arachidonate metabolism in aortas and platelets in aging rats. Prostaglandins Leuko Med 12:149, 1983.
29.
Henricksson, P., Hamberg, M. and Diczfalusy, U. Formationof 15-HETE as a major hydroxyeicosatetraenoic acid in atherosclerotic vessel wall. Biochim Biophys Acta 834:272, 1985.
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Stenson, W.F., Nickells, M.W. and Atkinson, J.P. Esterification of monohydroxy fatty acids into the lipids of a macrophage cell line. Prostaglandins 26:253, 1983.
33.
Clubb, F.J., Stegner, J.G., Fields, H.A., Willerson, J.T. and Buja, L.M. Vascular changes in the young Watanabe Heritable Hyperlipidemic (WHHL) rabbit. Fed Proc (ABSTRACT)45:812, 1986.
34.
Butler, M.M., Clubb, F.J., Stegner, J.G., Willerson, J.T. and Buja, L.M. Surface distribution of leukocytes and foam cells in young Watanabe Heritable Hyperlipidemic (WHHL) rabbit aortas. Fed. Proc. {Abstract) 46:977, 1987.
35.
Mathur, S.N., Field, F.J., Spector, A.A. and Armstrong, M.L. Increased production of lipoxygenase products by cholesterol-rich macrophages. Biochim Biophys Acta 83__Z7:13, 1985.
Rigaud, M., Razaka, G., Daret, D., Demond-Henri, J. and Formation of monohydroxyeicosatetraenoic acids from arachiby cultured rabbit aortic smooth muscle cells. Biochem Commun 11___22:242, 1983.
Editor: G. Kaley
532
Received: 11-23-87
Accepted: 8-18-88
OCTOBER 1988 VOL. 36 NO. 4