AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 288: H2210-H2218, 2005. First published December 30, 2004; doi:10.1152/ajpheart.00704.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/5/H2210    most recent
00704.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, W.
Right arrow Articles by Lee, H.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, W.
Right arrow Articles by Lee, H.-C.

Impaired arachidonic acid-mediated dilation of small mesenteric arteries in Zucker diabetic fatty rats

Wei Zhou,1 Xiao-Li Wang,1 Terry L. Kaduce,2 Arthur A. Spector,2 and Hon-Chi Lee1

1Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota; and 2Department of Biochemistry, University of Iowa, Iowa City, Iowa

Submitted 15 July 2004 ; accepted in final form 22 December 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Arachidonic acid (AA) is a precursor of important vasoactive metabolites, but the role of AA-mediated vasodilation in Type 2 diabetes is not known. Using Zucker diabetic fatty (ZDF) rats, we examined the effects of AA in small mesenteric arteries preconstricted with endothelin. In ZDF rat mesenteric arteries, 1 µM AA produced only one-third the amount of dilation as in vessels from lean control animals. In lean control rats, the effect of AA was significantly and predominantly inhibited by the lipoxygenase inhibitors baicalein and cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC). However, baicalein and CDC had no effect on AA-mediated dilation in ZDF rat mesenteric arteries. The major [3H]AA metabolite produced by isolated mesenteric arteries in both lean and ZDF rats was 12-hydroxyeicosatetraenoic acid (12-HETE), but the amount of [3H]12-HETE produced by ZDF rat vessels was only 36% of that of control vessels. In addition, 12-HETE produced similar amounts of dilation in lean and ZDF rat mesenteric arteries. Immunoblot analysis showed an 81% reduction in 12-lipoxygenase protein in ZDF rat mesenteric arteries. Immunofluorescence labeling showed strong nitrotyrosine signals in ZDF rat mesenteric arteries that colocalized with 12-lipoxygenase in endothelium, and 12-lipoxygenase coprecipitation with anti-nitrotyrosine antibodies was enhanced in ZDF rat vessels. We conclude that AA-mediated relaxation in ZDF rat small mesenteric arteries is impaired due to reduced 12-lipoxygenase protein and activity. Increased oxidative stress and nitration of 12-lipoxygenase may underlie the impairment of AA-mediated relaxation in small mesenteric arteries of diabetic rats.

Type 2 diabetes; 12-hydroxyeicosatetraenoic acid; lipoxygenase; cinnamyl-3,4-dihydroxy-cyanocinnamate; baicalein

ARACHIDONIC ACID (AA) IS THE most abundant and important precursor of vasoactive eicosanoids. Free AA is converted by 1) cyclooxygenase into prostaglandins and thromboxanes; 2) lipoxygenase into leukotrienes, lipoxins, and intrachain hydroxyeicosatetraenoic acids (HETEs); and 3) cytochrome P-450 monooxygenase into epoxyeicosatrienoic acids and chain-terminal HETEs (10). These products are important regulators of vasoreactivity. AA produces potent dilation in human coronary arterioles that is dependent on the cytochrome P-450 pathway (24), whereas the dilation produced in rat mesenteric microvessels is mediated mainly through the lipoxygenase pathway (23).

Type 2 diabetes has become a disease of epidemic proportions and is known to be associated with abnormal regulation of blood vessels (43). Type 2 diabetes-associated vascular dysfunction has been extensively studied in humans and various animal models (7, 19). Endothelial dysfunction with impaired activity of various endothelium-derived factors plays an integral role in Type 2 diabetes (7). The Zucker diabetic fatty (ZDF) rat is a useful animal model for Type 2 diabetes, although it has certain limitations (20) including slight hyperglycemia and higher serum insulin levels in lean control animals as compared with Sprague-Dawley rats (18). It has been reported (14) that in obese Zucker rats, NO-dependent flow-induced arteriolar dilation was attenuated with impaired arteriolar ability to regulate wall shear rate, and these changes were associated with increases in vascular superoxide levels. In addition, these animals have altered skeletal muscle arteriolar reactivity including increased myogenic activation (13) and {alpha}-adrenergic vasoconstriction (35). Recently, NO- and K+-channel-mediated dilator responses in basilar arteries were found (8) to be greatly diminished as a result of enhanced oxidative stress and PKC activation. It appears that reactive oxygen species play a central role in the pathogenesis of vasculopathy in Type 2 diabetes. In addition to alterations in pharmacological responses, reduced microvessel density and arteriolar distensibility and remodeling of the microcirculation also contribute substantially to impaired vascular function in diabetes (12).

AA is converted to potent vasodilator substances in the endothelium including prostacyclin, endothelium-derived hyperpolarizing factors, and 12-HETE (3, 9, 23, 24, 44). However, the role of AA in the regulation of vasoreactivity in Type 2 diabetes is not clear. The goal of this study was to examine AA-mediated relaxation in lean and ZDF rat small mesenteric arteries to determine the metabolic pathways mediating the effects of AA and the mechanism of abnormal AA-mediated vasodilation in ZDF rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male ZDF rats (ZDF/Gmi-fa/fa) and their age-matched genetic controls were obtained from Charles River Laboratories at 6–8 wk of age. All rats received Purina 3008 modified mouse/rat diet and were studied after 4 wk of hyperglycemia (>300 mg/dl) in ZDF rats according to a protocol approved by the Animal Use Committee of the Mayo Foundation.

Vasoreactivity measurements. Animals were anesthetized with pentobarbital sodium (50 mg/kg). The bowels and mesentery were rapidly excised and placed in ice-cold Krebs solution that contained (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.1 dextrose. Reactivity of isolated small mesenteric arteries (100–250 µm in diameter) was determined by video microscopy (23, 44). All vessels were equilibrated for at least 30 min in Krebs solution at 37°C before the start of the experiment.

To study the endothelium-dependent and -independent components of vasorelaxation, endothelium was removed by passing an air bubble through the isolated vessels. Vessels were used only if they did not relax with administration of acetylcholine (10–4 M) but had normal responses to nitroprusside (10–4 M) and KCl (60 mM).

Pharmacological interventions. All compounds were added to the circulating bath, and the cumulative dose responses were determined at 3- to 5-min intervals between doses. Vessels were constricted to ~40% of baseline diameter using endothelin-1, and the effects of AA (1 x 10–10 to 3 x 10–5 M) on vasorelaxation were examined. Vessels were discarded if they failed to produce 85% relaxation with 100 µM sodium nitroprusside or 30% constriction with 60 mM KCl.

To identify the metabolic pathways responsible for mediating the effects of AA, small mesenteric arteries were preincubated for 30 min with indomethacin (10–5 M) to inhibit the cyclooxygenase pathway, baicalein (10–6 M) or cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 10–6 M) to inhibit the 12-lipoxygenase pathway, or miconazole (10–5 M) to inhibit the cytochrome P-450 pathway.

To determine the ionic mechanism through which AA mediates its vasodilatory effects, vessels were preincubated for 30 min with iberiotoxin (IBTX; 10–7 M) to block the large-conductance Ca2+-activated K+ (BKCa) channels or 4-aminopyridine (4-AP; 1 mM) to block the voltage-gated K+ (Kv) channels.

Eicosanoid production in small mesenteric arteries of lean and ZDF rats. Small mesenteric arteries from two lean and two ZDF rats were dissected and collected in 1 ml of modified DMEM that contained 0.1 µM BSA (DMEM-BSA) on ice. The vessels were incubated for 1 h at 37°C with 1 ml of DMEM-BSA that contained 5 µM [5,6,8,9,11,12,14,15-3H]AA (1 µCi/nmol). Incubations were terminated by removing the media and extracting the lipids. Radioactive metabolites of AA were separated by reverse-phase high-performance liquid chromatography (44). The elution profile consisted of water and formic acid at pH 4.0 and an acetonitrile gradient that increased from 30 to 57% over 30 min, increased from 57 to 65% over 20 min, remained at 65% for 5 min, and then increased to 100% acetonitrile for 6 min. Radioactivity of the column effluent was measured by an in-line flow scintillation detector. Retention times of the radiolabeled components were compared with those of standards.

Immunoblot analysis. Isolated mesenteric arteries were homogenized in ~300 µl of T-PER tissue protein-extraction reagent (Pierce), resolved on a 4–15% Tris·HCl polyacrylamide gradient gel, and transferred electrophoretically onto a polyvinylidene difluoride membrane. Membranes were reversibly stained with Ponceau S, blotted against rabbit anti-murine 12-lipoxygenase antibodies (1:500 dilution; Cayman) and against anti-actin antibodies, and detected with Immun-Star horseradish peroxidase reagents (Bio-Rad). The relative amounts of lipoxygenase were determined by densitometric analysis, and the results were normalized against the amounts of actin.

RNA isolation and cDNA synthesis. Total RNA from small mesenteric arteries was isolated using an RNeasy Mini Kit (Qiagen) and treated with DNase I. RNAs were reverse transcribed using the ThermoScript RT-PCR System (Invitrogen) according to the manufacturer's instructions.

Quantitative real-time PCR. Real-time semiquantitative PCR analyses were performed using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Primers for the platelet-type and leukocyte-type 12-lipoxygenase and for the reference gene (ribosomal protein L32) were selected from published sequences (Table 1). With the use of these primers, PCR products of the expected sizes and no other products were amplified (data not shown). Duplicate samples were analyzed, and quantitative results were obtained by normalizing the target signal to the L32 signal (2, 41).


View this table:
[in this window]
[in a new window]
  		Table 1. Sequences of primers used for real-time RT-PCR

 
Immunohistochemical analysis. Frozen sections of mesenteric arteries were fixed with cold methanol for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min. After sections were blocked with 10% goat serum in PBS for 60 min, they were incubated with rabbit anti-murine 12-lipoxygenase antibodies (1:100 dilution; Cayman) and monoclonal anti-nitrotyrosine antibodies (1:70 dilution; Upstate) for 1.5 h and then with goat anti-mouse-fluorescein- and goat anti-rabbit-Texas red-conjugated secondary antibodies for 1 h. Sections were mounted in ProLong antifade reagent (Molecular Probes) and visualized using an LSM-510 confocal laser-scanning microscope (Carl Zeiss).

Immunoprecipitation. Mesenteric artery homogenates were precleared by a 2-h incubation with 0.25 µg of normal mouse IgG and 20 µl of protein G-agarose. Precleared supernatants were incubated with 4 µg of monoclonal anti-nitrotyrosine antibodies at 4°C overnight and then with protein G-agarose (30 µl) for 2 h. The immune complexes were collected by centrifugation at 2,000 g for 5 min, resolved by SDS-PAGE, and blotted against anti-12-lipoxygenase antibodies.

Statistical analysis. Data were analyzed using one-way ANOVA with post hoc Tukey test and are presented as means ± SE. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
At the time of the experiment, the ZDF rats were significantly heavier (412 ± 4 vs. 371 ± 4 g, n = 29; P < 0.0001) and more hyperglycemic (493 ± 9 vs. 164 ± 8 mg/dl, n = 29; P < 0.0001) than age-matched lean control rats.

Vasoactive effects of AA. AA produced dose-dependent relaxation in small mesenteric arteries from lean rats. At a 1 µM concentration, AA produced 54.3 ± 5.4% relaxation, and at 30 µM, AA produced 88.5 ± 5.1% relaxation (n = 12), which is similar to previous reports (23). In ZDF rat preparations, 1 µM AA produced only 18.6 ± 3.6% relaxation and 30 µM produced 51.2 ± 4.2% relaxation (n = 11; P < 0.001 vs. lean rats for both; Fig. 1A). Most of the AA-mediated relaxation was lost with removal of the endothelium in lean vessels but not in ZDF rat vessels. Nitroprusside (10–4 M) produced similar relaxation in lean and ZDF rat vessels (Fig. 1B). These results indicate that AA-mediated relaxation is impaired in small mesenteric arteries of ZDF rats.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 1. Relaxation in lean and Zucker diabetic fatty (ZDF) rat small mesenteric arteries was measured with arachidonic acid (AA) in vessels with ({blacksquare} and {bullet}) and without ({square} and {circ}) endothelium (A); with sodium nitroprusside (B); and with AA after 30 min of preincubation with 10–5 M indomethacin or miconazole (C and E), or with 10–6 M baicalein or cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC), baicalein + indomethacin (B+I), or miconzole + baicalein + indomethacin (M+B+I; D and F). *P < 0.05 vs. control or baseline.

 
In lean rats, incubation with miconazole or indomethacin had no significant effect on vasorelaxation produced by AA at concentrations of ≤3 µM (Fig. 1C). In contrast, after incubation with baicalein, AA-mediated vasorelaxation was inhibited at all concentrations. Incubation with baicalein plus indomethacin had no additional inhibitory effects, and incubation with all three inhibitors together also had no additional inhibitory effects except at 30 µM of AA (Fig. 1D). Inhibition of the lipoxygenase pathway with another inhibitor, CDC (1 µM), produced results similar to those with baicalein (Fig. 1, D and F). These results suggest that AA-mediated mesenteric relaxation occurs predominantly through metabolites produced by the lipoxygenase pathway. The cyclooxygenase and cytochrome P-450 epoxygenase pathways may also participate in AA-mediated vasorelaxation but only in the presence of high AA concentrations. Incubation with the pathway inhibitors in ZDF rat vessels did not affect AA-mediated vasorelaxation except at the highest AA dose with baicalein and miconazole treatments (Fig. 1, E and F), which suggests that the production of vasoactive AA metabolites formed by the lipoxygenase pathway might be impaired.

Role of K+ channels on AA-mediated vasorelaxation. Incubation of small mesenteric arteries from lean rats with IBTX produced a significant reduction in relaxation at AA concentrations ≥10–8 M with only 29.1 ± 4.6% relaxation at 1 µM AA and 56.5 ± 4.3% relaxation at 30 µM AA (P < 0.001 for both vs. baseline; Fig. 2A). In contrast, incubation of ZDF rat vessels with IBTX had no effect on AA-mediated relaxation except at 30 µM (Fig. 2B). Similarly, 4-AP produced a significant reduction in mesenteric relaxation in lean rats at all AA concentrations tested, with 23.9 ± 3.2% relaxation at 1 µM AA and 58.8 ± 4.9% relaxation at 30 µM AA (P < 0.005 for both vs. baseline; Fig. 2C). However, 4-AP did not alter AA-mediated relaxation in ZDF rat vessels (Fig. 2D). These results suggest that AA has lost its ability to produce mesenteric relaxation through activation of BKCa and Kv channels in ZDF rats.



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. Effects of K+ channel blockers on AA-mediated relaxation in small mesenteric arteries from lean (A and C) and ZDF (B and D) rats. AA-mediated relaxation was measured after 30 min of preincubation with 10–7 M iberiotoxin (IBTX; A and B) or 1 mM 4-aminopyridine (4-AP; C and D). *P < 0.05 vs. baseline.

 
Eicosanoid production by small mesenteric arteries. The main radiolabeled metabolite produced by lean and ZDF rat mesenteric arteries was 12-HETE (retention time, 42 min; Fig. 3). The ZDF rat vessels produced only 36% as much radiolabeled 12-HETE as the lean rat vessels (12-HETE produced: lean, 903.4; ZDF, 331.5 pmol/g of wet wt; total wet weight of mesenteric arteries from two rats: lean, 115.9; ZDF, 125.8 mg). ZDF rat vessels also showed a reduction in the radiolabeled component with a retention time of 30 min, which is identical to the 12-HETE {beta}-oxidation product that was previously identified as 8-hydroxyhexadecatrienoic acid [(8-OH)16:3] in endothelial and vascular smooth muscle cells (16, 27). Thus the lean rat mesenteric arteries converted 7% of the available AA to 12-HETE, whereas the ZDF rat mesenteric arteries converted only 3.4%. These results demonstrate that conversion to 12-HETE, a lipoxygenase product, is the major pathway for AA metabolism in small mesenteric arteries in lean rats, and the production of this lipoxygenase product is impaired in ZDF rats.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 3. AA metabolites produced by small mesenteric arteries from lean (A) and ZDF (B) rats. Isolated vessels were incubated with 5 x 10–6 M [3H]AA (specific activity, 1 µCi/nmol) for 1 h at 37°C. Lipids were extracted and analyzed by reverse-phase HPLC. Major peak at 42 min had the same retention time as a 12-hydroxyeicosatetraenoic acid (12-HETE) standard. Peak at 30 min was identified previously as 8-hydroxyhexadecatrienoic acid, a {beta}-oxidation product of 12-HETE.

 
Response of small mesenteric arteries to 12-HETE. Both lean and ZDF rat vessels had similar dose-dependent relaxation responses on exposure to 12-HETE (Fig. 4A). The effects of 12-HETE were significantly attenuated by IBTX and 4-AP in both lean and ZDF rat vessels (Fig. 4, B and C), which suggests that both BKCa and Kv channels mediate the 12-HETE effects. These results indicate that impaired AA-mediated relaxation in ZDF rat small mesenteric arteries is not due to a reduced response of the vessels to 12-HETE.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Effects of 12-HETE (1 x 10–10 to 1 x 10–6 M) with or without IBTX and 4-AP on relaxation of small mesenteric arteries from lean (A and B) and ZDF (A and C) rats. *P < 0.05 vs. baseline.

 
Expression of 12-lipoxygenase. Immunoblot analysis showed reduced 12-lipoxygenase protein expression in ZDF rat mesenteric arteries (Fig. 5A, top). This difference was not due to the amount of protein loaded (Fig. 5A, bottom). The relative amount of 12-lipoxygenase protein was reduced by 81% in ZDF rat mesenteric arteries compared with lean rat vessels (1.55 ± 0.15 in lean vs. 0.30 ± 0.04 in ZDF rats, n = 4; P < 0.005; Fig. 5B), which indicates that impaired AA-mediated relaxation in ZDF rat mesenteric arteries is due to reduced amounts of 12-lipoxygenase protein. Because this antibody cross-reacts with both platelet- and leukocyte-type 12-lipoxygenase standards (Fig. 5C), we are not able to distinguish between them immunologically.



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 5. Immunoblot analysis of 12-lipoxygenase (12-LOX) in small mesenteric arteries from lean and ZDF rats. Immunoblot using anti-12-lipoxygenase antibodies (75-kDa band; A, top). Same membrane is shown, but blotted against anti-actin antibodies (A, bottom). Group data from four pairs of lean and ZDF rats (B). Densitometric measurements of 12-lipoxygenase were normalized against those of actin. Immunoblot using anti-12-lipoxygenase antibodies against porcine leukocyte and human platelet 12-lipoxygenase standards and rat aorta homogenate (included as reference control; C). *P < 0.05 vs. lean.

 
We then measured 12-lipoxygenase mRNA levels of both platelet and leukocyte types in lean and ZDF rat mesenteric arteries. The mRNA levels for the platelet-type 12-lipoxygenase were not significantly different between lean and ZDF rat mesenteric arteries (Fig. 6A). However, the leukocyte-type 12-lipoxygenase mRNA levels were 4.6 ± 1.0-fold higher in ZDF than lean control rat mesenteric arteries (n = 9; P = 0.002 vs. lean control; Fig. 6B). The platelet-type 12-lipoxygenase mRNA level in lean rat vessels was fourfold higher than the leukocyte-type counterpart (n = 9; P < 0.005; Fig. 6C), but the platelet- and leukocyte-type 12-lipoxygenase mRNAs were equally expressed in ZDF rat mesenteric arteries (Fig. 6D). These results indicate that expression of the 12-lipoxygenase gene was not suppressed in ZDF rat vessels. Rather, the reduction in 12-lipoxygenase protein in ZDF rat vessels was due to either impaired translation or increased turnover. We also found that ZDF rat vessels had a 4.19 ± 0.99-fold increase in 5-lipoxygenase mRNA compared with lean rat vessels (n = 8; P < 0.05), which suggests that 5-lipoxygenase gene expression also was activated and not suppressed in ZDF rat vessels.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6. Relative abundance of 12-lipoxygenase mRNA in small mesenteric arteries from lean and ZDF rats. Mesenteric mRNA was isolated from nine pairs of lean and ZDF rats, and both platelet- and leukocyte-type 12-lipoxygenase mRNAs were measured using real-time PCR techniques. Results are expressed as means ± SE showing the relative abundances of the platelet-type 12-lipoxygenase mRNA between lean and ZDF rat vessels (A), the leukocyte-type 12-lipoxygenase mRNA between lean and ZDF rat vessels (B), the platelet- and leukocyte-type 12-lipoxygenase mRNAs in lean rat vessels (C), and the platelet- and leukocyte-type 12-lipoxygenase mRNAs in ZDF rat vessels (D).

 
Tyrosine nitration of 12-lipoxygenase. Both lean and ZDF rat mesenteric arteries were strongly labeled in Texas red with anti-12-lipoxygenase antibodies (Fig. 7A). In contrast, the fluorescein signals of anti-nitrotyrosine antibodies were very weak in lean rat vessels but strong in ZDF rat vessels (Fig. 7B). Merging the Texas red and the fluorescein images produced strikingly strong yellow signals in endothelium of ZDF rat vessels (Fig. 7C) but not from lean control rat vessels. These results suggest that ZDF vessels have higher levels of protein tyrosine nitration, and 12-lipoxygenase in the ZDF vessel is strongly tyrosine nitrated, especially in endothelium.



View larger version (82K):
[in this window]
[in a new window]
  		Fig. 7. Tyrosine nitration of 12-lipoxygenase. Vessel sections were double-labeled with rabbit anti-murine 12-lipoxygenase (A) and monoclonal anti-nitrotyrosine (B) antibodies and were then incubated with goat-anti-rabbit IgG antibodies conjugated with Texas red and goat anti-mouse IgG antibodies conjugated with fluorescein. Areas of 12-lipoxygenase and nitrotyrosine colocalization are shown in yellow in the merged images (C).

 
Immunoprecipitation experiments showed that more 12-lipoxygenase is coprecipitated with an anti-nitrotyrosine antibody in ZDF than in lean rat preparations, which again suggests that 12-lipoxygenase in ZDF rat vessels is more strongly tyrosine nitrated (Fig. 8). This finding is especially significant considering that the amount of 12-lipoxygenase protein in ZDF rat mesenteric arteries is only one-third of that in lean control animals, and it confirms the results of immunofluorescence labeling.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 8. Western blotting analysis of 12-lipoxygenase immunoprecipitated with tyrosine nitration. Lean and ZDF rat mesenteric artery homogenates were immunoprecipitated with anti-nitrotyrosine monoclonal antibodies, and the immune complexes were analyzed by immunoblot with anti-12-lipoxygenase antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We found that small mesenteric arteries in rats with Type 2 diabetes have impaired AA-mediated relaxation. This impairment is due to reduced production of 12-HETE, a potent vasoactive lipoxygenase metabolite of AA. The reduced 12-HETE production in turn is due to reduced 12-lipoxygenase enzyme protein in ZDF rat mesenteric arteries. However, lipoxygenase mRNA levels were not reduced in ZDF rats, which suggests that there might be increased inactivation and degradation of lipoxygenase due to the observed increase in tyrosine nitration.

We found that AA is a potent dilator of small mesenteric arteries in lean rats, and this effect was dependent on lipoxygenase activity. These results are in agreement with those reported by Miller et al. (23). Likewise, 12-HETE formed by the lipoxygenase pathway produced vasodilation in porcine coronary microvessels (44). However, in human coronary microvessels, the effects of AA were mediated via a cytochrome P-450 epoxygenase product (24), which suggests that the metabolic fate of AA varies between species and vascular beds.

AA-mediated relaxation is significantly impaired in ZDF rats. In ZDF rat mesenteric arteries, 1 µM AA generated only 34% of the relaxation seen in lean control rats. Blockade of 12-lipoxygenase resulted in significant reduction of the effects of AA in lean but not ZDF rat mesenteric arteries, which suggests that the lipoxygenase metabolite is the major contributor to AA-mediated relaxation in lean rats. Our failure to observe any increase in the formation of cyclooxygenase or cytochrome P-450 products when ZDF rat small mesenteric arteries were incubated with [3H]AA indicates that the reduced lipoxygenase activity was not associated with a compensatory increase of activity in the other two pathways.

Demonstration that ZDF rat mesenteric arteries responded normally to 12-HETE suggests that the impaired AA response was due to a deficiency in lipoxygenase activity that resulted in reduced production of vasoactive metabolites. This was first confirmed by biochemical analysis of radiolabeled AA products formed by isolated mesenteric artery segments. Indeed, AA is mainly converted to 12-HETE by vessels from lean rats, but ZDF rat vessels only produced one-third as much. This was further confirmed by immunoblot analysis, which demonstrated that ZDF rat mesenteric arteries contain only 19% of the amount of lipoxygenase protein as lean rat vessels. Hence, the reduced lipoxygenase activity could be attributed to the reduced lipoxygenase protein in ZDF rats.

Real-time RT-PCR indicated that the small mesenteric arteries of lean rats expressed both platelet- and leukocyte-type 12-lipoxygenase, but the platelet type was the predominant form. This is consistent with previous findings that the 12-HETE produced by mouse brain cerebral microvessels, rat basilar arteries, and porcine coronary microvessels is the S-enantiomer (9, 26, 44), the stereoisomer produced by the platelet enzyme. The reduced levels of lipoxygenase in mesenteric arteries, however, are not due to downregulation of lipoxygenase transcription. In fact, mRNA levels of platelet-type 12-lipoxygenase were unchanged, and those of leukocyte-type 12-lipoxygenase were elevated four- to fivefold in ZDF rats. Platelet 12-lipoxygenase deficiencies have been reported in myeloproliferative disorders (31). Although platelet 12-lipoxygenase is activated in some forms of malignancies such as prostate cancer (38), it is suppressed in others such as cervical cancer (29). The upregulation of leukocyte-type 12-lipoxygenase mRNA expression that occurred in ZDF rat vessels could be induced by hyperglycemia (28) or by cytokines and other stress signals associated with diabetes (4). Recently, db/db diabetic mice were found (17) to have increased urinary 12-HETE excretion and increased 12/15-lipoxygenase expression in endothelial cells isolated from these animals. Results from human studies are mixed. Reduced 12-lipoxygenase activity has been observed in platelets of patients with non-insulin-dependent diabetes (39), but an increase in urinary 12-HETE in female diabetic patients also has been reported (36). However, whole body 12-lipoxygenase product measurements may not reflect what is occurring in small mesenteric arteries. Reduced 12-HETE production in mesenteric arteries of ZDF rats may account for the blunted vasodilator responses of the mesenteric microvasculature in diabetes.

In addition, our results indicate that at high concentrations of AA (≥10 µM), cyclooxygenase and cytochrome P-450 pathways also contribute to AA-mediated mesenteric relaxation in lean rats (see Fig. 1C). Products of these pathways are important modulators of vascular reactivity. The major cyclooxygenase product of AA in the vascular endothelium is PGI2, a powerful vasodilator and inhibitor of platelet aggregation (25). PGI2 produces autocrine and paracrine effects by activating the IP prostanoid receptor, which leads to an increase in intracellular cAMP (1). Abnormalities in PGI2 regulation have been reported in diabetes. For example, PGI2-induced vasorelaxation in mesentery artery is impaired in Type 1 diabetes (37); serum PGI2 levels are decreased in Type 2 diabetic patients with angiopathy (22); and endothelial dysfunction in diabetic rats can be overcome by oral administration of a PGI2 analog. The defective PGI2-dependent vasodilator response in obese Zucker rats is due to PGI2 inactivation by superoxide anion (11), and an increase in superoxide and protein kinase C (PKC) activity in the basilar arteries of obese Zucker rats causes impaired PGI2-dependent vasodilation and cerebrovascular dysfunction (8). Recently, decreased cytochrome P-450 epoxygenase and increased epoxide hydrolase expression were also observed in the mesenteric arteries of obese Zucker rats (42). Therefore, all three AA metabolic pathways involved in vascular regulation have the propensity to become abnormal in diabetes and may contribute to impairment of vasodilator function. However, in the ZDF rat mesenteric arteries that we studied, the abnormality in the lipoxygenase pathway appears to have a greater effect under ordinary conditions, although all three pathways contribute to impaired AA-mediated vasorelaxation at high AA concentrations.

Oxidative stress is increased and may account for the vascular dysfunction that occurs in diabetes (15). In obese Zucker rats, basal plasma levels of isoprostane 8-epi-PGF2{alpha}, an in vivo marker of lipid peroxidation, were elevated by fivefold and markedly reduced by dietary vitamin E (21). As indicated by the superoxide-sensitive dye hydroethidine, production of superoxide was found to be significantly increased in basilar arteries of obese compared with lean Zucker rats (8). Increased superoxide production facilitates its reaction with NO to form peroxynitrite. This reaction would not only inactivate and decrease the bioavailability of NO but would also produce a highly potent oxidant and nitrating agent that has an array of biological actions that contribute to endothelial dysfunction (32). Detection of nitrotyrosine in proteins is considered to be a biomarker for endogenous peroxynitrite formation (6, 30, 40). Hyperglycemia has been shown to promote tyrosine nitration via peroxynitrite, producing inactivation of prostacyclin synthase in human endothelial cells (5). Aortic segments from obese Zucker rats showed significantly more nitrotyrosine staining (increased by 51%) than lean Zucker rat aorta (13). We found that mesenteric arteries from ZDF rats had high levels of protein tyrosine nitration, and the strong nitrotyrosine signals were colocalized with 12-lipoxygenase in endothelium. Furthermore, 12-lipoxygenase was coprecipitated with anti-nitrotyrosine immune complexes, which suggests that 12-lipoxygenase in ZDF rat mesenteric arteries is a substrate of tyrosine nitration by peroxynitrite. The most common consequence of protein tyrosine nitration is loss of function (40), and this could contribute to the reduced 12-lipoxygenase activity in ZDF rat mesenteric arteries. In addition, protein tyrosine nitration has been shown to enhance susceptibility to proteolytic degradation (33) and may cause increased 12-lipoxygenase turnover, which would lead to reduced enzyme protein in ZDF rat mesenteric arteries. Our findings add to the growing list of proteins that are modified by tyrosine nitration, which contributes to the progression of cardiovascular diseases (40). These novel observations suggest that enhanced oxidative stress could be a common denominator for the mechanism of vascular and endothelial dysfunction in diabetes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by National Heart, Lung, and Blood Institute Grants HL-63754, HL-72845, and HL-74180.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H.-C. Lee, Div. of Cardiovascular Diseases, Dept. of Internal Medicine, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: Lee.honchi{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Breyer RM, Bagdassarian CK, Myers SA, and Breyer MD. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 41: 661–690, 2001.[CrossRef][ISI][Medline]
  2. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29: 23–39, 2002.[Abstract]
  3. Campbell WB and Gauthier KM. What is new in endothelium-derived hyperpolarizing factors? Curr Opin Nephrol Hypertens 11: 177–183, 2002.[CrossRef][ISI][Medline]
  4. Chang WC. Cell signaling and gene regulation of human 12(S)-lipoxygenase expression. Prostaglandins Other Lipid Mediat 71: 277–285, 2003.[CrossRef][Medline]
  5. Cosentino F, Eto M, De Paolis P, van der Loo B, Bachschmid M, Ullrich V, Kouroedov A, Delli Gatti C, Joch H, Volpe M, and Luscher TF. High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothelial cells: role of protein kinase C and reactive oxygen species. Circulation 107: 1017–1023, 2003.[Abstract/Free Full Text]
  6. Crow JP and Ischiropoulos H. Detection and quantitation of nitrotyrosine residues in proteins: in vivo marker of peroxynitrite. Methods Enzymol 269: 185–194, 1996.[ISI][Medline]
  7. De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, and Vanhoutte PM. Endothelial dysfunction in diabetes. Br J Pharmacol 130: 963–974, 2000.[CrossRef][ISI][Medline]
  8. Erdos B, Snipes JA, Miller AW, and Busija DW. Cerebrovascular dysfunction in Zucker obese rats is mediated by oxidative stress and protein kinase C. Diabetes 53: 1352–1359, 2004.[Abstract/Free Full Text]
  9. Faraci FM, Sobey CG, Chrissobolis S, Lund DD, Heistad DD, and Weintraub NL. Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K+ channels. Am J Physiol Regul Integr Comp Physiol 281: R246–R253, 2001.[Abstract/Free Full Text]
  10. Foegh ML and Pamwell PW. The eicosanoids: prostaglandins, thromboxanes, leukotrienes, and related compounds. In: Basic and Clinical Pharmacology, edited by Katzung BG. New York: McGraw-Hill, 2002.
  11. Frisbee JC. Impaired dilation of skeletal muscle microvessels to reduced oxygen tension in diabetic obese Zucker rats. Am J Physiol Heart Circ Physiol 281: H1568–H1574, 2001.[Abstract/Free Full Text]
  12. Frisbee JC. Remodeling of the skeletal muscle microcirculation increases resistance to perfusion in obese Zucker rats. Am J Physiol Heart Circ Physiol 285: H104–H111, 2003.[Abstract/Free Full Text]
  13. Frisbee JC, Maier KG, and Stepp DW. Oxidant stress-induced increase in myogenic activation of skeletal muscle resistance arteries in obese Zucker rats. Am J Physiol Heart Circ Physiol 283: H2160–H2168, 2002.[Abstract/Free Full Text]
  14. Frisbee JC and Stepp DW. Impaired NO-dependent dilation of skeletal muscle arterioles in hypertensive diabetic obese Zucker rats. Am J Physiol Heart Circ Physiol 281: H1304–H1311, 2001.[Abstract/Free Full Text]
  15. Griendling KK and FitzGerald GA. Oxidative stress and cardiovascular injury. Part II: animal and human studies. Circulation 108: 2034–2040, 2003.[Free Full Text]
  16. Hadjiagapiou C, Sprecher H, Kaduce TL, Figard PH, and Spector AA. Formation of 8-hydroxyhexadecatrienoic acid by vascular smooth muscle cells. Prostaglandins 34: 579–589, 1987.[CrossRef][Medline]
  17. Hatley ME, Srinivasan S, Reilly KB, Bolick DT, and Hedrick CC. Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice. J Biol Chem 278: 25369–25375, 2003.[Abstract/Free Full Text]
  18. Jordan JE, Simandle SA, Tulbert CD, Busija DW, and Miller AW. Fructose-fed rats are protected against ischemia/reperfusion injury. J Pharmacol Exp Ther 307: 1007–1011, 2003.[Abstract/Free Full Text]
  19. Kirpichnikov D and Sowers JR. Diabetes mellitus and diabetes-associated vascular disease. Trends Endocrinol Metab 12: 225–230, 2001.[CrossRef][Medline]
  20. Laight DW, Anggard EE, and Carrier MJ. Investigation of basal endothelial function in the obese Zucker rat in vitro. Gen Pharmacol 35: 303–309, 2002.
  21. Laight DW, Kengatharan KM, Gopaul NK, Anggard EE, and Carrier MJ. Investigation of oxidant stress and vasodepression to glyceryl trinitrate in the obese Zucker rat in vivo. Br J Pharmacol 125: 895–901, 1998.[CrossRef][ISI][Medline]
  22. Migdalis IN, Kalogeropoulou K, Iliopoulou V, Triantafilou P, Charalabides J, Mortzos G, and Cordopatis C. Plasma levels of endothelin, lipid peroxides and prostacyclin in diabetic patients with macroangiopathy. Diabetes Res Clin Pract 54: 129–136, 2001.[CrossRef][ISI][Medline]
  23. Miller AW, Katakam PV, Lee HC, Tulbert CD, Busija DW, and Weintraub NL. Arachidonic acid-induced vasodilation of rat small mesenteric arteries is lipoxygenase-dependent. J Pharmacol Exp Ther 304: 139–144, 2003.[Abstract/Free Full Text]
  24. Miura H and Gutterman DD. Human coronary arteriolar dilation to arachidonic acid depends on cytochrome P-450 monooxygenase and Ca2+-activated K+ channels. Circ Res 83: 501–507, 1998.[Abstract/Free Full Text]
  25. Moncada S, Higgs EA, and Vane JR. Human arterial and venous tissues generate prostacyclin (prostaglandin x), a potent inhibitor of platelet aggregation. Lancet 1: 18–20, 1977.[CrossRef][ISI][Medline]
  26. Moore SA, Giordano MJ, Kim HY, Salem N Jr, and Spector AA. Brain microvessel 12-hydroxyeicosatetraenoic acid is the (S) enantiomer and is lipoxygenase derived. J Neurochem 57: 922–929, 1991.[CrossRef][ISI][Medline]
  27. Moore SA, Prokuski LJ, Figard PH, Spector AA, and Hart MN. Murine cerebral microvascular endothelium incorporate and metabolize 12-hydroxyeicosatetraenoic acid. J Cell Physiol 137: 75–85, 1988.[CrossRef][Medline]
  28. Natarajan R, Gerrity RG, Gu JL, Lanting L, Thomas L, and Nadler JL. Role of 12-lipoxygenase and oxidant stress in hyperglycaemia-induced acceleration of atherosclerosis in a diabetic pig model. Diabetologia 45: 125–133, 2002.[CrossRef][ISI][Medline]
  29. Nigam S, Kumar GS, Sutherland M, Schewe T, Ikawa H, Yamasaki Y, Ueda N, and Yamamoto S. Metabolic suppression of platelet-type 12-lipoxygenase in human uterine cervix with invasive carcinoma. Int J Cancer 82: 827–831, 1999.[Medline]
  30. Reiter CD, Teng RJ, and Beckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 275: 32460–32466, 2000.[Abstract/Free Full Text]
  31. Schafer AI. Deficiency of platelet lipoxygenase activity in myeloproliferative disorders. N Engl J Med 306: 381–386, 1982.[Abstract]
  32. Schopfer FJ, Baker PRS, and Freeman BA. NO-dependent protein nitration: a cell signaling event or an oxidative inflammatory response? Trends Biochem Sci 28: 646–654, 2003.[CrossRef][ISI][Medline]
  33. Souza JM, Choi I, Chen Q, Weisse M, Daikhin E, Yudkoff M, Obin M, Ara J, Horwitz J, and Ischiropoulos H. Proteolytic degradation of tyrosine nitrated proteins. Arch Biochem Biophys 380: 360–366, 2000.[CrossRef][ISI][Medline]
  34. Spector AA, Gordon JA, and Moore SA. Hydroxyeicosatetraenoic acids (HETEs). Prog Lipid Res 27: 271–323, 1988.[CrossRef][ISI][Medline]
  35. Stepp DW and Frisbee JC. Augmented adrenergic vasoconstriction in hypertensive diabetic obese Zucker rats. Am J Physiol Heart Circ Physiol 282: H816–H820, 2002.[Abstract/Free Full Text]
  36. Suzuki N, Hishinuma T, Saga T, Sato J, Toyota T, Goto J, and Mizugaki M. Determination of urinary 12(S)-hydroxyeicosatetraenoic acid by liquid chromatography-tandem mass spectrometry with column-switching technique: sex difference in healthy volunteers and patients with diabetes mellitus. J Chromatogr B Analyt Technol Biomed Life Sci 783: 383–389, 2003.[Medline]
  37. Taylor PD and Poston L. The effect of hyperglycaemia on function of rat isolated mesenteric resistance artery. Br J Pharmacol 113: 801–808, 1994.[ISI][Medline]
  38. Timar J, Raso E, Dome B, Li L, Grignon D, Nie D, Honn KV, and Hagmann W. Expression, subcellular localization and putative function of platelet-type 12-lipoxygenase in human prostate cancer cell lines of different metastatic potential. Int J Cancer 87: 37–43, 2000.[CrossRef][ISI][Medline]
  39. Tohjima T, Honda N, Mochizuki K, Kinoshita J, Watanabe K, Arisaka T, Kawamori R, Nakamura M, Kurahashi Y, Yoshimoto T, and Yamamoto S. Decreased activity of arachidonate 12-lipoxygenase in platelets of Japanese patients with non-insulin-dependent diabetes mellitus. Metabolism 47: 257–263, 1998.[Medline]
  40. Turko IV and Murad F. Protein nitration in cardiovascular diseases. Pharmacol Rev 54: 619–634, 2002.[Abstract/Free Full Text]
  41. Ueno S, Yamada H, Moriyama T, Honda K, Takano Y, Kamiya HO, and Katsuragi T. Measurement of dorsal root ganglion P2X mRNA by SYBR Green fluorescence. Brain Res Brain Res Protoc 10: 95–101, 2002.[CrossRef][Medline]
  42. Zhao X, Dey A, Romanko OP, Stepp DW, Wang MH, Zhou Y, Jin L, Poloock JS, Webb RC, and Imig JD. Decreased epoxygenase and increased epoxide hydrolase expression in the mesenteric artery of obese Zucker rats. Am J Physiol Regul Integr Comp Physiol 288: R188–R196, 2005.[Abstract/Free Full Text]
  43. Zimmet P, Alberti KG, and Shaw J. Global and societal implications of the diabetes epidemic. Nature 414: 782–787, 2001.[CrossRef][Medline]
  44. Zink MH, Oltman CL, Lu T, Katakam PV, Kaduce TL, Lee H, Dellsperger KC, Spector AA, Myers PR, and Weintraub NL. 12-Lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation. Am J Physiol Heart Circ Physiol 280: H693–H704, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Circ. Res.Home page
T. Lu, T. He, Z. S. Katusic, and H.-C. Lee
Molecular Mechanisms Mediating Inhibition of Human Large Conductance Ca2+-Activated K+ Channels by High Glucose
Circ. Res., September 15, 2006; 99(6): 607 - 616.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. Ye, W. Zhou, T. Lu, S. G. Jagadeesh, J. R. Falck, and H.-C. Lee
Mechanism of rat mesenteric arterial KATP channel activation by 14,15-epoxyeicosatrienoic acid
Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1326 - H1336.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
T. Lu, X.-L. Wang, T. He, W. Zhou, T. L. Kaduce, Z. S. Katusic, A. A. Spector, and H.-C. Lee
Impaired Arachidonic Acid-Mediated Activation of Large-Conductance Ca2+-Activated K+ Channels in Coronary Arterial Smooth Muscle Cells in Zucker Diabetic Fatty Rats
Diabetes, July 1, 2005; 54(7): 2155 - 2163.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/5/H2210    most recent
00704.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, W.
Right arrow Articles by Lee, H.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, W.
Right arrow Articles by Lee, H.-C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.