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Department of Biomedical Sciences and The Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211; and Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L
Submitted 24 July 2003 ; accepted in final form 12 November 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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nitric oxide; prostacyclin; endothelial nitric oxide synthase; endothelium-independent relaxation; vascular smooth muscle
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Ninety-one adult, male Yucatan miniature swine, 8–12 mo of age, 25–40 kg body wt (Charles River, Maine and Sinclair Research Farm, Columbia, MO), were used with protocols approved by the Animal Care and Use Committee at the University of Missouri. Pigs were housed in rooms maintained at 20–23°C with a 12:12-h light-dark cycle. One-half of the pigs were provided a normal-fat (NF) diet (Purina Lab Mini-pig Chow; 8% of daily caloric intake derived from fat), and one-half of the pigs were fed a high-fat (HF) diet (46% of their daily caloric intake from fat) consisting of pig chow supplemented with cholesterol (2.0%), coconut oil (17.1%), corn oil (2.3%), and sodium cholate (0.7%) (5, 32, 39). Pigs were exercise trained (Ex) or remained sedentary (Sed) for 16–20 wk, beginning 4 wk after dietary intervention was initiated. Pigs continued to consume the HF or NF diet during the entire protocol. The experimental design used four groups of pigs: 1) NF-Sed, 2) NF-Ex, 3) HF-Sed, and 4) HF-Ex. Plasma lipid data from the pigs used in this study have been reported previously (32). The HF diet induced elevated plasma cholesterol (NF-Sed = 59 ± 4 mg/dl, HF-Sed = 404 ± 44 mg/dl), triglyceride (NF-Sed = 30 ± 6 mg/dl, HF-Sed = 40 ± 4 mg/dl), HDL-C (NF-Sed = 32 ± 1 mg/dl, HF-Sed = 96 ± 6 mg/dl), and LDL-C (NF-Sed = 24 ± 3 mg/dl, HF-Sed = 232 ± 23 mg/dl) in HF pigs. Importantly, Ex did not significantly alter the lipid profiles of these pigs (32).
Training Program
All pigs were familiarized with running on a motorized treadmill and randomly assigned to Ex or Sed groups for 16–20 wk. The Ex group completed the 16- to 20-wk endurance training program described previously (14, 22). Pigs assigned to the Sed group were restricted to their enclosures (2 x 4 m pens) and did not exercise.
Vascular Ring Preparation
At the end of the protocol, pigs were sedated with ketamine (30 mg/kg im) and anesthetized with pentobarbital sodium (35 mg/kg iv). Segments of the left anterior descending coronary artery (LAD) were (isolated from the same location in all pigs) removed and trimmed of connective tissue and fat. Axial length, inside diameter (ID), and outside diameter (OD) of each vascular ring were measured with a Filar calibrated micrometer eye piece. Vasomotor reactivity was examined with the rings stretched to the length that produced maximal active tension (Lmax), as described previously (12, 19, 25, 26).
Experimental Design
Direct and interactive effects of HF and Ex on contractile responses, endothelium-independent and endothelium-dependent responses (EDR), were examined. Because there are reports that HF diets produce impaired EDR in response to some, but not all, endothelium-dependent agonists (13, 30), whereas others report generalized endothelial dysfunction (6, 21, 24), we determined whether HF produced generalized or selective endothelial dysfunction in adult male pigs by examining responses to three different endothelium-dependent agonists [aggregating platelets, 5-HT, and bradykinin (BK)]. Three different protocols were used.
Vascular reactivity protocol 1. Protocol 1 examined relaxation responses produced by 5-HT, aggregating platelets, and sodium nitroprusside (SNP). One intact and one denuded LAD ring were treated with 30 µM PGF2
to precontract the vascular rings for examination of relaxation responses. Rings were treated throughout the experiment with indomethacin (Indo; 5 µM) to block production of products of cyclooxygenase (COX) and ketanserin tartrate (1 x 10-6 M) to block smooth-muscle 5-HT2 receptors (13, 30). Concentration-response relationships for vasodilators were determined by cumulative addition of 5-HT (10-10 to 10-5 M) and SNP (10-10 to 10-4 M), in half-log increments to the bath (26). Concentration-response relationships for aggregating platelets were determined by cumulative addition of platelet-rich solution (25, 50, 75, and 100 x 103 platelets/µl) to the bath (13, 30, 31). Platelets were harvested from 300 ml of autologous blood collected before removal of the heart and prepared as described (13, 30, 31). For denuded LAD experiments, the endothelium was removed by gentle rubbing of the luminal surface with fine-tipped forceps and confirmed when the ring produced <5% relaxation in response to 10-6 M BK (26). Endothelium dependency of responses was evaluated by comparing responses of intact and denuded rings. SNP responses were examined after all other dose-response relationships were completed.
Vascular reactivity protocol 2. Protocol 2 was designed to examine contractile responses produced by KCl, 5-HT, and aggregating platelets (in the absence of Indo and ketanserin tartrate). This experiment was designed to determine the importance of smooth-muscle 5-HT2-serotonergic receptor and second-messenger system, interactions between the effects of 5-HT on smooth muscle and endothelium in vascular dysfunction caused by HF, and the effects of Ex on these processes. Concentration-response relationships were determined by cumulative addition of KCl (20–100 M), 5-HT (10-10 to 10-5 M), and aggregating platelets (cumulative addition of platelet-rich solution: 25, 50, 75, and 100 x 103 platelets/µl) to the bath (13, 30, 31).
Vascular reactivity protocol 3. The third protocol examined the relative contributions of NO, prostacyclin (PGI2), and/or non-COX and non-NOS pathways by using procedures outlined in detail previously (39, 40). Four LAD rings were obtained from each pig: one untreated control, one treated with 0.3 mM NG-nitro-L-arginine methyl ester (L-NAME) to block NO release from NOS, one treated with 5 µM Indo to block COX, and one treated with both blockers (L-NAME + Indo) to evaluate the role of endothelium-derived hyperpolarizing factor (EDHF) and/or other endothelium-derived mediators. All four rings were precontracted with PGF2 (30 µM), and EDR was assessed by using ACh (10-10 to 10-4 M) and BK (10-11 to 10-6 M). One subset of animals underwent measures of coronary structure and function in the cardiac catheterization laboratory before isolation of coronary arteries, as outlined above. There was no effect of these procedures on responses of isolated arteries, so the data are pooled.
Solutions and Drugs
Krebs bicarbonate buffer solution contained (in mM) 131.5 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 11.2 glucose, 20.8 NaHCO3, 0.003 propranolol, and 0.025 EDTA. Solutions were aerated with 95% O2-5% CO2 (pH 7.4) and maintained at 37°C. All drugs and chemicals were purchased from Sigma Chemical.
Immunohistochemistry
Samples of LAD were dissected and immersed in 10% formalin for a minimum of 24 h, embedded routinely in paraffin, and sectioned serially at 5-µm thickness. Immunohistochemistry was performed, as reported previously (16, 29, 39), by using mouse monoclonal antibodies against scavenger receptor A (SRA-E5 1:100, Cosmo Bio), eNOS (1:800, BD Transduction Laboratories), rabbit polyclonal antibodies against caveolin-1 (Cav-1; 1:200, Santa Cruz Laboratories), SOD-1 (1:800, Stressgen Biotechnology), and SOD-2 (1:800, Stressgen Biotechnology). For negative controls, histological sections were prepared without primary antibodies. Sections were examined and photographed with an Olympus BX40 photomicroscope.
Immunoblot Analysis
LAD rings were homogenized in a buffer consisting of 50 mM Tris·HCl, pH 7.4, 6 M urea, and 2% SDS by using a ground-glass homogenizer. After a 2-h incubation at 45°C and centrifugation (10,000 g, 1 min), protein concentration of the supernatants was determined by using the bicinchoninic acid assay (Pierce). Before electrophoresis, aliquots of these samples were supplemented with 150 mM dithiothreitol and boiled for 1 min. Gels were loaded with 30 µg of protein, electrophoresed, and electroblotted to polyvinylidene difluoride membranes. Membranes were analyzed for eNOS and Cav-1 (Transduction Laboratories, 1:3,500 and 1:2,000, respectively) and SOD-1 and SOD-2 (Stressgen, both 1:2,000). Secondary antibodies were conjugated with horseradish peroxidase. Antibody and blocking solutions contained 5% nonfat milk and 0.1% Tween in Tris-buffered saline. Chemiluminescent (Amersham) signals were captured and quantified by using the Syngene Chemi-Genus2 Bio-Imaging System and software (Syngene).
Statistical Analysis
All values are means ± SE. Concentration-response curves were evaluated by using mixed-factor, repeated-measures ANOVA. Data were analyzed with each animal counted as one observation for comparisons between groups with respect to each vasoactive agent or combination of agents. Data for the contractile responses were analyzed as developed tension (grams of tension) and as specific tension. Specific tension was calculated by dividing developed tension by the cross-sectional area of vessel wall (OD - ID x length). EC50 was defined as the vasodilator concentration that produced 50% inhibition of a PGF2 preconstriction. EC50 was determined for each arterial ring with linear interpolation between the log concentrations that produced responses just below and above 50%. Significance of differences between mean values of citrate synthase activity, heart weight-to-body weight ratio, endurance times during treadmill performance test, protein content (immunoblot results), maximal effect, and EC50 values were assessed with one-way ANOVA. P values of <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Vascular Ring Characteristics
LADs from HF animals had similar ODs, but smaller IDs, than LADs from NF pigs (Table 1). There were no statistically significant differences in LAD wall thickness among groups. Axial length, resting tension at Lmax, and percent stretch to Lmax were not significantly altered by diet, exercise, or the diet x exercise interaction.
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Contractile Responses
KCl, ACh, and 5-HT. LADs from HF pigs developed significantly more force in response to increasing doses of KCl than NF LADs (Fig. 1A). Training did not alter KCl responses in LADs from pigs on either diet. As reported previously (25), ACh did not induce EDR, but caused increases in developed tension that were similar in LADs from all four groups (Fig. 1B). Treatment of the rings with L-NAME, Indo, and L-NAME + Indo did not significantly alter responses to ACh in any group (data not shown). 5-HT elicited a dose-dependent increase in developed tension in LADs from all groups of pigs (Fig. 2A). Contraction induced by high doses of 5-HT tended to be greater in LADs from HF pigs (P = 0.06). There was not a significant effect of Ex on contractile responses to 5-HT.
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Aggregating platelets and PGF2. Aggregating platelets elicited a dose-dependent increase in developed tension in intact LADs from all groups of pigs (Fig. 3A). Mixed-factor, repeated-measures ANOVA revealed a significant main effect of diet, indicating that contraction induced by aggregating platelets was greater in LADs from pigs fed the HF diet while Ex had no effect on responses to aggregating platelets. We also noted in results from protocol 3 that LADs from HF-Ex pigs often exhibited greater developed tension during preconstriction with 30 µM PGF2 than those from NF groups [PGF2 developed tension (in g): NF-Sed = 6.5 ± 1.1, NF-Ex = 6.7 ± 0.8, HF-Sed = 5.9 ± 1.2, HF-Ex = 9.3 ± 0.8]. ANOVA revealed that this difference was not statistically significant (P = 0.09).
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Relaxation Responses
5-HT. In the presence of Indo and ketanserin, 5-HT elicited a concentration-dependent relaxation of LADs (Fig. 2B). Mixed-factor, repeated-measures ANOVA revealed a significant main effect of diet (P = 0.03), indicating that 5-HT-induced relaxation was impaired in LAD from pigs fed the HF diet. Furthermore, statistical analysis revealed a significant diet x Ex interaction (P = 0.05), indicating that 5-HT-induced relaxation was impaired more in HF-Ex than in HF-Sed.
Aggregating platelets. In all groups, responses of the LADs to aggregating platelets were changed from contraction (Fig. 3A) to relaxation by treatment with Indo and ketanserin tartrate (Fig. 3B), with the relaxation in the NF LADs being greater than relaxation observed in the HF LADs. Relaxation to aggregating platelets was not altered by Ex. There was no longer a difference between relaxation to aggregating platelets in NF and HF groups after denudation (Fig. 3C).
BK. BK elicited a dose-dependent relaxation of LAD rings from all groups of pigs (Fig. 4). Mixed-factor, repeated-measures ANOVA did not reveal a significant main effect of diet on BK-induced relaxation. However, there was a significant diet x exercise x dose interaction (P = 0.04), indicating that relaxation produced by intermediate and high doses of BK was less in LADs from HF-Sed pigs. BK-induced relaxation was improved in HF-Ex LADs such that relaxation was not significantly different from that in NF-Sed or NF-Ex LADs.
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The effects of L-NAME treatment on BK-induced relaxation are summarized in Fig. 5. Mixed-factor ANOVA revealed a significant main effect of L-NAME (Fig. 5), indicating that NOS inhibition decreased BK-induced relaxation. ANOVA did not reveal a significant L-NAME x diet interaction, indicating that L-NAME treatment had similar effects on BK-induced relaxation in NF and HF arteries (Fig. 5, A and B). Also, statistical analysis did not reveal a significant L-NAME x Ex interaction, indicating that L-NAME treatment had similar effects in LADs from Sed and Ex pigs (Fig. 5, C and D). The percent BK-induced relaxation inhibited by L-NAME treatment was 36% in HF-Ex and 17% in HF-Sed (Table 2), suggesting that Ex increased the importance of NOS-generated NO in HF LADs.
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Treatment with Indo did not significantly alter responses to BK in any group (Table 2). L-NAME + Indo inhibited BK-induced relaxation in NF-Sed, NF-Ex, and HF-Ex LADs such that relaxation was no longer greater than that in HF-Sed arteries (Fig. 6A). Exercise did not alter the effects of double blockade on BK-induced relaxation in NF pigs (Fig. 6B), and L-NAME + Indo had similar effects on LADs from HF-Ex and NF-Ex pigs (Fig. 6C). Interestingly, in HF-Sed arteries, BK-induced relaxation tended to be greater in the presence of Indo than in untreated LADs (Fig. 6D), and L-NAME + Indo increased relaxation (42 vs. 32%, Table 2) compared with relaxation with L-NAME alone (17%, Table 2). BK-induced relaxation was similar in NF-Sed, NF-Ex, and HF-Ex arteries after treatment with L-NAME alone and treatment with both L-NAME + Indo (Table 2). These observations suggest involvement of an Indo-sensitive constrictor in HF-Sed LAD. Importantly, results also suggest that the Indo-sensitive constrictor substance is no longer evident in LADs from HF-Ex pigs, i.e., Ex decreases or prevents production of this vasoconstrictor in HF-Ex LADs. Consistent with this interpretation, mixed-factor, repeated-measures ANOVA revealed an interaction between L-NAME + Indo and exercise such that L-NAME + Indo produced a greater decrease in BK-induced relaxation in HF-Ex than in HF-Sed LADs (Fig. 7A), whereas Indo treatment alone had no effect on BK-induced relaxation in either HF-Sed or HF-Ex (Fig. 7B).
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SNP Responses
LADs from all four groups exhibited a dose-dependent relaxation in response to SNP. Neither HF nor Ex altered LAD responses to SNP (Table 2).
Immunoblot Analysis
Immunoblot analyses of LAD ring homogenates revealed that eNOS protein content was similar in all groups (Fig. 8A). ANOVA revealed a main effect of diet on Cav-1 protein content, indicating that HF diet produced an increase in Cav-1 content (Fig. 8B). SOD-1 protein content was lower in NF-Ex and HF-Sed than in NF-Sed (Fig. 8C). SOD-2 protein content was lower in NF-Ex and HF-Sed than in HF-Ex (Fig. 8D). Ex appeared to increase SOD-1 and SOD-2 protein content in LADs of HF animals, because SOD-1 and SOD-2 content in HF-Ex LADs was not different from that in NF-Sed.
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Immunohistochemistry
There was positive immunoreactivity for eNOS in the endothelium of all groups. Importantly, faint positive staining also was present in macrophage foam cells in HF-Sed and HF-Ex pigs (Fig. 9). Positive immunoreactivity for Cav-1, SOD-1, and SOD-2 was present in the endothelium and smooth muscle in all groups of pigs (Figs. 10, 11, 12). Staining for SOD-2 was punctate and consistent with staining of mitochondria (Fig. 12). Foam cells in LADs from HF-Ex stained well for SOD-1, whereas those in LADs from HF-Sed did not (Fig. 11). Foam cells were identified in the intima of LADs from both HF groups by staining for SRA (Fig. 13). There was no positive SRA macrophage staining in LADs from NF groups (Fig. 13). In the absence of primary antibody against eNOS, SOD-1, SOD-2, Cav-1, or SRA, no immunoreactivity was detected (data not shown).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Influence of HF Diet on EDR
Role of NO release and NO bioactivity. HF produced endothelial dysfunction in the LADs of these male, adult pigs (Figs. 3 and 4). Our results provide insight into the potential role of the following mechanisms for decreased EDR (8): decreased eNOS expression and protein content, decreased NOS activity, decreased NO bioavailability, decreased production of PGI2, decreased production of EDHF or other endothelium-derived relaxing factors, and increased production of constrictor substances.
The percent EDR inhibited by L-NAME treatment was less in the HF-Sed LADs (17%) than in LADs from NF-Sed and NF-Ex pigs (27 and 34%) (Table 2), indicating that the contribution of NO release to EDR was decreased by HF, consistent with our hypothesis. However, HF did not decrease eNOS protein expression, as we had proposed. Immunoblot results indicate similar eNOS protein content in LADs from all groups (Fig. 8). Interestingly, this HF diet increased eNOS protein content of LADs from HF-Sed female pigs (39). Faint eNOS-positive staining of macrophage foam cells was revealed by immunohistochemical analysis of LADs from both HF-Sed and HF-Ex male pigs (Fig. 9), similar to eNOS staining in foam cells of LADs from female pigs on this HF diet (39). This suggests the possibility that endothelial cell eNOS content may have been decreased by HF, even though total eNOS content of the arteries was not decreased because of eNOS in other cells. We did not anticipate that other vascular cells would express eNOS protein in HF LADs.
We examined Cav-1 expression in our arteries to evaluate the possibility that NO-mediated EDR of HF LADs was impaired due to increased expression of Cav-1 (8, 9) as hypercholesterolemia increases Cav-1 expression in cultured endothelial cells (7). Consistent with this hypothesis, our results reveal a main effect of diet, indicating that Cav-1 protein content was increased by HF (Fig. 8). However, immunohistochemical results suggest that both endothelial and smooth-muscle cells of HF LADs have more Cav-1 than do those of low-fat-diet pigs (Fig. 10). Therefore, whereas our results are consistent with the notion that EDR is decreased in HF due to decreased activity of eNOS, resulting from increased Cav-1 and eNOS-Cav-1 binding (7–9), it will be necessary to quantify Cav-1 content specifically in the endothelial cells to be sure. Of interest, this HF diet did not produce increased LAD Cav-1 content in female pigs (39).
Decreased maximal BK-induced relaxation without significant changes in sensitivity in HF-Sed LADs (Table 2) is consistent with a decrease in NO bioavailability resulting from increased reactive oxygen species (ROS). SOD-1 content was less in HF-Sed LADs than in NF-Sed LADs (Fig. 8). Decreased SOD-1 content in HF-Sed LADs may be the result of low expression of SOD-1 in foam cells in HF-LADs (Fig. 11). The observation of SRA-positive macrophage foam cells in HF-LADs in addition to endothelial and smooth-muscle cells is another important difference between HF LADs and NF LADs (Fig. 13). Macrophage foam cells from atherosclerotic rabbit aorta have been shown to produce superoxide, NO, and hydrogen peroxide (27). Thus immunoblot and immunohistochemical results are consistent with a contribution of increased ROS in HF LADs, causing reduced NO-mediated EDR (34). These results are also different from those reported in female pigs on this diet (39). Female pig LADs exhibited no change in SOD-1 among groups but did exhibit increased SOD-2 in response to HF.
In summary, our results indicate that NOS-mediated, BK-induced relaxation is decreased in LADs of male pigs on this HF diet. The immunoblot and immunohistochemical results are consistent with the notion that NO-mediated relaxation is decreased due to the actions of ROS and suggest that not all of the eNOS protein in the HF arteries is located in endothelial cells. We conclude that decreased NO-mediated relaxation contributes to blunted EDR in LADs from adult male HF-Sed pigs.
Role of PGI2 and other non-NOS mechanisms. Indo treatment increased BK-induced relaxation only in HF-Sed LADs, suggesting the presence of an Indo-sensitive vasoconstrictor. Furthermore, L-NAME alone and L-NAME + Indo had similar inhibitory effects on EDR in arteries from NF-Sed, NF-Ex, and HF-Ex pigs. In contrast, BK-induced relaxation was not inhibited by treatment with L-NAME + Indo in HF-Sed LADs (Fig. 6D). These results indicate an Indo-sensitive vasoconstrictor substance present in HF-Sed LADs.
Finally, because responses to BK were similar in all groups during treatment with L-NAME + Indo (Fig. 6A), it does not appear that BK-induced relaxation in HF-Sed LADs was reduced because of decreased release of EDHF or other non-NOS, non-COX vasodilator mechanisms. Therefore, we conclude that blunted EDR observed in LADs of HF-Sed male pigs is the result of two mechanisms: 1) production of an Indo-sensitive vasoconstrictor substance; and 2) decreased NO-mediated relaxation resulting from decreased SOD-1 expression and increased Cav-1 content.
Influence of Exercise Training on EDR
Role of NO release and NO bioactivity. Ex improved BK-induced relaxation of LADs from HF-Ex (Fig. 4), but not EDR stimulated by aggregating platelets or 5-HT. The relatively small magnitude of EDR responses to aggregating platelets and 5-HT, compared with EDR produced by BK, may explain the lack of effect of Ex on these responses.
Because L-NAME treatment caused 36% inhibition of BK-induced EDR in HF-Ex LADs (Table 2) vs. only 17% in HF-Sed LADs (Table 2), results indicate that Ex in HF pigs increased the importance of NO, generated by NOS to EDR. Although there was not a significant interaction between L-NAME treatment and exercise (P = 0.1) (Fig. 5D), L-NAME did decrease maximal BK-induced relaxation in HF-Ex LADs, but not HF-Sed LADs (Table 2), also suggesting an increased contribution of NOS to EDR after Ex. This exercise-induced increased NO-mediated relaxation of NO was not the result of increased eNOS protein content or decreased Cav-1 protein content of HF-Ex LADs (Fig. 8B). However, SOD-1 and SOD-2 content were greater in HF-Ex LADs than in HF-SEDs, indicating that improved NO-mediated relaxation in LADs from HF-Ex pigs is, in part, the result of improved NO bioavailability (Fig. 8, C and D).
The increased SOD-1 content in HF-Ex vs. HF-Sed LADs (Fig. 8C) is consistent with previous observations that Ex increased SOD-1 expression in coronary arterioles (28) and in aortic endothelial cells of female Yucatan pigs trained with this same training program (29). It is interesting that Ex decreased SOD-1 content in LAD of male pigs on the NF diet (Fig. 8C), but increased SOD-1 content in NF-Ex female pigs, relative to NF-SEDs (39).
Role of PGI2 and other non-NOS mechanisms. The protective effect of exercise on EDR in HF LADs does not appear to be the result of enhanced release of PGI2 because Indo did not significantly alter BK-induced relaxation in NF-Ex or HF-Ex LADs (Fig. 7). The Indo-sensitive constrictor substance in HF-Sed LADs, discussed above, was not evident in the LADs of HF-Ex pigs. Ex decreased or prevented production of this vasoconstrictor substance. Consistent with this interpretation, statistical analysis revealed an interaction between double blockade and exercise, indicating that L-NAME + Indo produces a greater decrease in BK-induced relaxation in HF-Ex than in HF-Sed (Fig. 7). During treatment with L-NAME + Indo, BK-induced EDR was similar in all groups (Fig. 7A), indicating that NOS- and COX-independent vasodilator mechanisms (EDHF) have similar effects in LADs from all groups of pigs. The observation that EDHF-mediated EDR was not altered by HF or Ex is similar to results from female pigs (39).
Because HF LADs tended to develop greater tension in response to several agonists, it is important to evaluate the potential role of greater tension on EDR in these arteries. PGF2 induced similar amounts of tension in HF-Sed and NF LADs. Therefore, blunted EDR in HF-Sed LADs is not likely, due to greater constriction of HF-Sed arteries. Similarly, the fact that HF-Ex exhibited 
20% greater PGF2-induced force (P = 0.09) argues against the possibility that HF-Ex LADs exhibited greater EDR in response to BK because of less preconstriction. Furthermore, the facts that EDR to aggregating platelets was similar in HF-Ex and HF-Sed LADs and that there were no differences among the BK-induced relaxations during double blockade or among the SNP responses of these arteries also argue that differences observed in BK-induced responses of these LADs are predominantly the result of differences in the endothelium.
Gender Perspectives
These results indicate that this HF diet has similar effects on EDR of coronary arteries of male and female pigs (39), except that LADs from HF-Sed males appeared to be more impaired than those from female pigs. Consistent with this sex difference, the immunohistochemistry results of this study also suggest that foam cells are more apparent in male LADs than in female pigs on the same diet (39). Thus the severity of endothelial dysfunction and perhaps the degree of vascular disease produced by this HF diet are influenced by sex, but the primary effects and mechanisms responsible for said effects appear similar between males and females. Thus, in both sexes, blunting of BK-induced EDR in coronary arteries was the result of a combination of decreased release and activity of NO and increased production of an Indo-sensitive constrictor. Also, the contributions of non-NOS and non-COX systems do not appear to be altered by HF or Ex in either sex of pig (39). Finally, Ex attenuated these deleterious effects of HF on endothelial function in male LADs by increasing NO-mediated EDR and by reducing the Indo-sensitive constrictor substance in a manner similar to exercise effects observed in female pigs on the same HF diet (39). These effects of exercise in HF males were not mediated by changes in eNOS protein content of the LADs. Interestingly, Ex and HF appeared to have different effects on Cav-1, SOD-1, and SOD-2 contents of male LADs (Fig. 8) than in female LADs (39). Thus there are some differences in adaptative mechanisms between male and female pigs exposed to HF and Ex, even though the net effect of these different adaptations produces similar effects on endothelial function.
Conclusions
Results of this study indicate that EDR was impaired by HF in coronary arteries of adult male pigs. The detrimental effects of HF were characterized by blunted EDR responses to BK, 5-HT, and aggregating platelets. BK-induced relaxation appeared to be blunted in HF-Sed LADs due to the presence of an Indo-sensitive vasoconstrictor substance and decreased NO-mediated relaxation. The decreased NO-mediated relaxation appears to be the result of decreased SOD content and increased Cav-1 content of the HF-Sed LADs. Results indicate that endurance Ex prevented or reversed the deleterious effects of hypercholesterolemia on BK-induced EDR. The Ex-induced preservation and/or improvement of endothelial function in HF-Ex arteries is partially the result of enhanced relaxation by NO (perhaps due to increased SOD content) and removal of an Indo-sensitive vasoconstrictor. Training did not appear to alter the role of non-NOS, non-COX factors such as EDHF.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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GRANTS
This work was supported by National Heart, Lung, and Blood Institute Grants HL-52490 and HL-36088 (to M. H. Laughlin), National Institute on Aging Grant AG-00988 (to C. R. Woodman), and Heart and Stroke Foundation of Ontario Grant NA-4604 (to J. W. E. Rush).
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FOOTNOTES |
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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.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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