Impairment of Coronary Blood Flow Regulation by... : Journal of Cardiovascular Pharmacology (original) (raw)
Vascular endothelium contributes to the control of both vascular tonus and platelet function (1). A major endothelium-derived relaxing factor is nitric oxide (NO), which is derived from L-arginine (L-ARG) (2,3). Endothelium-derived nitric oxide (EDNO) is a potent vasodilator and inhibitor of platelet adhesion and aggregation in in vitro studies and regulates coronary blood flow (CBF) in in vivo studies (4-6).
Diabetes mellitus is a major cause of ischemic coronary heart disease. Endothelial dysfunction has been implicated in the pathogenesis of diabetic vascular disease (7). Endothelium-dependent relaxation is reported to be impaired in the aorta and in pial arterioles in experimental diabetic models (8-10). Endothelium-dependent relaxation was shown to be abnormal in forearm resistance vessels of patients with insulin-dependent diabetic mellitus (11,12). The substances suggested to be related to the mechanism of impaired endothelium-dependent relaxation in diabetes include the vasoconstrictor prostanoid or prostanoids, superoxide radicals, and advanced glycation endproducts (13-16).
We examined whether CBF regulation by EDNO is impaired in the diabetic state. For this purpose, we compared effects of acetylcholine (ACh), an endothelium-dependent relaxant agent, and adenosine (Ado), an endothelium-independent relaxant agent, on CBF in vehicle-treated dogs and dogs with alloxan-induced diabetes.
MATERIALS AND METHODS
Thirty-three healthy adult mongrel dogs weighing 13-17 kg were randomly divided into two groups: 21 dogs were treated with a single intravenous (i.v.) injection of alloxan monohydrate (40 mg/kg) (diabetic dogs), and the other 12 received normal saline (vehicle dogs). These dogs were monitored every week for body weight and blood sugar level, which was determined with a electrochemistry shuffle dextrometer (ExacTech 2, Medisense, U.S.A.). The dogs received standard dogfood containing 1.56% L-ARG and water ad libitum. Four weeks after injection of alloxan or saline, all dogs were anesthetized with intravenous sodium pentobarbital (30 mg/kg body weight) and ventilated with a mixture of room air and 100% O2 by an artificial respirator. The arterial blood pH, PO2, and PCO2 were maintained within physiological range. A catheter was inserted in the right axillary artery for measurement of the arterial pressure. The heart was exposed by a left thoracotomy at the fifth intercostal space, and the proximal portion of the left circumflex coronary artery (LCX) was dissected free from the surrounding tissue before its first marginal branch. Heparin (200-U/kg bolus injection followed by 1,000-U injection every 60 min) and aspirin (17-mg/kg bolus injection) were administered intravenously to inhibit platelet aggregation and vasoactive prostaglandins. In our preliminary study, this dose of aspirin decreased the flow increment by intracoronary infusion of arachidonic acid (375 μg) from 132 ± 23 to 9 ± 4 ml/100 g. The LCX was cannulated and perfused with the arterial blood from the left common carotid artery through an extracorporeal bypass tube. Our previous study in which we used this coronary bypass system showed that the decrease in coronary perfusion pressure was negligible as compared with the aortic pressure (17). An electromagnetic flow probe (Nihon Kohden FF-045F, Tokyo, Japan) was placed in the middle of the bypass tube. The LCX blood flow was continuously measured with a flowmeter (Nihon Kohden MFV3200) and recorded with a polygraph (Fukuda Denshi MIC-9800, Japan) together with the arterial pressure and two ECG leads (I and II). Left ventricular pressure (LVP) was also measured from the cannula inserted in the left ventricle through the apical dimple. Drugs (ACh, Ado, adenosine, and _N_G-nitro-L-arginine methyl ester (L-NAME) were infused into the LCX from the middle portion of the bypass tube. All experiments were performed in accordance with the guidelines on experimental animals issued by Kumamoto University School of medicine and were approved by the Center of Laboratory Animals.
Drug preparation
Alloxan (Sigma Chemical, St. Louis, MO, U.S.A.) was dissolved with warm sterile saline. ACh chloride (ACh, Daiichi Seiyaku, Tokyo, Japan) was dissolved in normal saline to produce concentrations of 100 ng/kg/ml, 300 ng/kg/ml, and 1 μg/kg/ml; 0.1 ml of these solutions (10, 30, and 100 ng/kg, respectively) was infused into the LCX. Ado (Sigma) was dissolved in normal saline to produce a concentration of 10 μg/kg/ml; 0.1 ml of this solution (Ado 1 μg/kg) was infused into the LCX. L-NAME (Sigma) was dissolved in normal saline whose volume was adjusted in each dog by the basal LCX blood flow to derive L-NAME concentrations of 10-5, 10-4 and 10-3_M_ in arterial blood when administered into the LCX at a rate of 0.5 ml/min. L-ARG (Sigma) and D-ARG (Wako Chemical, Osaka, Japan) were dissolved in normal warm saline. Cu, Znsuperoxide dismutase (Cu, Zn-SOD, Wako) was dissolved in normal saline.
Protocols
In both vehicle and diabetic dogs, systemic hemodynamic parameters were recorded after a 30-min stabilization period. ACh (10, 30, and 100 ng/kg) and Ado (1 μg/kg) were infused into the LCX for 8 s at a constant rate, and their effect on the LCX blood flow was observed. Each drug was administered after hemodynamic parameters had returned to the baseline values. To analyze blood chemistry and plasma levels of L-ARG, L-citrulline, L-glutamine, and L-lysine, blood was drawn from the left ventricle. The dogs were assigned to one of the following three experiments.
- Experiment 1: Effects of L-NAME on ACh- and Ado- induced flow increments In 6 dogs with alloxan-induced diabetes and 6 vehicle dogs, three doses of L-NAME (10-5, 10-4, and 10-3_M_ in LCX blood) were cumulatively administered into the LCX. Each dose was infused for 6 min at a rate of 0.5 ml/min with a syringe pump. The effects of ACh (100 ng/kg) and Ado (1 μg/kg) were examined before and after each dose of L-NAME.
- Experiment 2: Effects of L- and D-ARG on ACh- and Ado-induced flow increments In 6 vehicle dogs and 6 dogs with alloxan-induced diabetes, L-ARG (150 mg/kg) was administered intravenously. Systemic hemodynamic parameters and the effects of ACh (10, 30, 100 ng/kg) and Ado (1 μg/kg) on CBF were observed before and 10 min after L-ARG administration. In 4 other dogs with alloxan-induced diabetes, D-ARG (150 mg/kg) was administered intravenously and systemic hemodynamic parameters and the effects of ACh and Ado were observed before and after D-ARG administration.
- Experiment 3: Effects of Cu, Zn-SOD on ACh- and Ado-induced flow increments in diabetic dogs In the remaining 5 dogs with alloxan-induced diabetes, Cu, Zn-SOD (10 ng/kg/min) was infused into the LCX for 10 min and the effects of ACh (10, 30, 100 ng/kg) and Ado (1 μg/kg) on CBF were observed.
At the end of the experiments, Evans blue was injected into the perfused segment area, and the animal was killed with an overdose of sodium pentobarbital. The heart was excised, and the weight of the perfused segment area was measured.
Data analysis
The effects of both ACh and Ado were expressed as the percent change in CBF and coronary vascular resistance (CVR) from the baseline values. CBF was expressed as blood flow/100 g myocardium in the perfused segment area. Coronary vascular resistance (CVR) was defined as the ratio of mean blood pressure (BP) to CBF. Rate-pressure product (RPP) was defined as heart rate (HR) × systolic BP (SBP). Plasma concentrations of L-ARG, L-citrulline, L-glutamine, and L-lysine were analyzed by high-performance liquid chromatography (18).
All data are mean ± SEM. Student's unpaired t test was used for comparison of the parameters between vehicle-treated and dogs with alloxan-induced diabetes. Student's paired t test was used for comparison of the parameters before and after L- or D-ARG and Cu, Zn-SOD administration. The coronary flow responses to ACh and Ado infusion and the changes in hemodynamic parameters after each dose of L-NAME infusion were assessed statistically by repeated-measures analysis of variance (ANOVA); if a significant change was detected by ANOVA, Fisher's protected least significant difference multiple comparison test was performed to assess which individual difference was statistically significant. The level of significance was p < 0.05.
RESULTS
Blood chemistry
Table 1 summarizes blood sugar levels and blood chemistry in vehicle-treated and diabetic dogs. Fasting blood sugar in diabetic dogs at the time of the experiment was significantly increased as compared with that in vehicle-treated dogs. Total cholesterol level in diabetic dogs was also higher than that in vehicle-treated dogs. Plasma concentration of either L-ARG or L-citrulline was not significantly different between vehicle-treated and diabetic dogs. However, the concentrations of both L-lysine and L-glutamine in diabetic dogs were significantly lower than those in vehicle-treated dogs. No other chemical parameter was different between the two groups.
Hemodynamic parameters
Neither basal CBF nor any other hemodynamic parameter was significantly different between vehicle-treated and diabetic dogs (Table 2).
Responses to ACh and Ado
The percent changes in CBF and CVR after ACh administration in diabetic dogs were significantly attenuated as compared with those in vehicle-treated dogs (Fig. 1). Neither percent change in CBF nor percent change in CVR after Ado was significantly different between vehicle-treated and diabetic dogs (Fig. 2).
Effects of cumulative doses of L-NAME in vehicle-treated and diabetic dogs
Hemodynamic parameters.Table 3 summarizes the effects of L-NAME on hemodynamic parameters in vehicle-treated and diabetic dogs. The total amount of L-NAME was not different between the two groups. CBF was significantly decreased and CVR was significantly increased dose dependently after infusion of L-NAME in vehicle-treated and diabetic dogs. Mean BP was significantly increased by cumulative L-NAME infusion in diabetic dogs. Other hemodynamic parameters did not change significantly after cumulative L-NAME infusion.
Responses to ACh and Ado. The percent changes in CBF and CVR after ACh administration were significantly attenuated by L-NAME in a dose-dependent manner in vehicle-treated and diabetic dogs. Before L-NAME infusion, the percent changes in CBF after ACh (100 ng/kg) in vehicle-treated and diabetic dogs were 184.2 ± 12.1 and 134.5 ± 10.6%, respectively (p < 0.05), and those in CVR were -63.8 ± 1.6 and -56.8 ± 2.4%, respectively (p < 0.05) (Fig. 3). After infusion of 10-3_M_ L-NAME, the percent changes in CBF induced by ACh in vehicle-treated and diabetic dogs were decreased to 98.1 ± 13.6 and 88.8 ± 8.5%, respectively, and those in CVR were -48.1 ± 2.9 and -45.0 ± 2.8%, respectively. After L-NAME infusion (10-3_M_), ACh responses (increases in CBF and decreases in CVR) were not statistically different between the two groups. Neither percent changes in CBF nor percent changes in CVR after Ado were affected by L-NAME in either vehicle-treated or diabetic dogs (Fig. 3).
Effects of L- and D-ARG
Hemodynamic parameters. In diabetic dogs, CBF was significantly increased, and both CVR and LV end-diastolic pressure (EDP) were significantly decreased after L-ARG (Table 4). After D-ARG administration, hemodynamic parameters did not change (Table 5). In vehicle-treated dogs, hemodynamic parameters did not change after L-ARG (Table 4).
Responses to ACh and Ado. In vehicle-treated dogs, neither percent change in CVR after ACh nor that after Ado administration was affected by L-ARG infusion (Fig. 4). In diabetic dogs, the percent change in CVR after ACh was significantly restored by L-ARG but not by D-ARG (Fig. 5). The percent changes in CVR after Ado were not affected by L- or D-ARG infusion.
Effects of Cu, Zn-SOD in dogs with alloxan-induced diabetes Cu, Zn-SOD did not significantly affect any systemic hemodynamic parameters (Table 5). Neither percent change in CVR after ACh nor that after Ado administration was affected by Cu, Zn-SOD (Fig. 5).
DISCUSSION
Impairment of ACh-Induced vasodilation in the diabetic state
No is a major factor in endothelium-dependent relaxation (2). It is synthesized by vascular endothelial cells from L-ARG (3). No synthesis was inhibited by L-NAME in a substrate-competitive manner in both in vitro and in vivo studies (5,19). Endothelium-dependent relaxation has been reported to be impaired in the aorta, corpus cavernosum tissue, and pial arterioles in experimental models of diabetes mellitus (8-10). ACh-induced flow increment is mediated by endothelium-derived NO (5,20) and hyperpolarizing factor or factors. In the present study, the effect of ACh was significantly impaired in diabetic dogs as compared with vehicle-treated dogs and, after the infusion of the maximum dose of L-NAME, no significant difference in the effect of ACh was noted between the two groups, indicating that CBF regulation by EDNO is impaired in the diabetic state. In our previous study, ACh-induced flow increment is not completely abolished by L-NAME (5). The remaining flow increment after the maximum dose of L-NAME is considered to be due to mechanism or mechanisms other than NO, such as hyperpolarizing factor. The mechanism of impaired endothelium-dependent relaxation in response to ACh in experimental diabetes was reported to be related to the production of cyclooxygenase constrictor substances, such as thromboxane A2(13). However, this was unlikely in the present study since aspirin, a cyclooxygenase inhibitor, was administered to inhibit the production of prostaglandins. Organ chamber experiments showed that endothelium-dependent relaxation in experimental diabetes was restored by exogenously administered SOD (14,15), which suggested glycation and inactivation of intrinsic Cu, Zn-SOD in diabetic mellitus and resultant production of superoxide radicals inactivating EDNO (21). However, in the present study, ACh-induced flow increment was not potentiated by Cu, Zn-SOD. In an in vivo study such as the present one, superoxide radicals may have been dismutated by other reducing agents even if their production was enhanced in diabetic dogs.
On the other hand, previous in vitro studies showed that the production of EDRF is increased in the diabetic state (22,23). We can not explain the conflicting findings in these previous and the present in vivo studies. It may be related to the use of different vasculatures, our use of aspirin for the inhibition of prostaglandins, and differences in the duration of diabetes and study design.
Responses to L-ARG
L-ARG is a major precursor of EDNO (3). In the present study, L- but not D-ARG restored the percent change in CVR after ACh administration in diabetic dogs. Taylor and Poston reported that L-ARG prevented the abnormality of ACh-induced relaxation in a hyperglycemic condition (24). They suggest that substrate availability for NO synthase is rate-limiting under hyperglycemic conditions. On the other hand, L-ARG did not potentiate the percent change in CVR after ACh administration in vehicle-treated dogs. This findings is in accordance with previously reported results (25,26) and, in normal conditions, the generation of NO from L-ARG is considered not to be rate-limiting.
Advanced glycation endoproducts represent the terminal adducts of the nonenzymatic glycation reaction between glucose and the amino group of protein (27). Lysine is considered the most important amino acid for generation of these proteins (28). In the present study, the plasma concentration of L-lysine was significantly decreased in diabetic dogs as compared with vehicle-treated dogs which may indicate that advanced glycation endoproducts were produced in our experimental model. Bucala and associates reported that advanced glycation endoproducts quenched NO activity in both in vitro and in vivo studies and that aminoguanidine prevented the NO quenching (16). Aminoguanidine is structurally similar to L-ARG in that these compounds contain two chemically equivalent guanidino nitrogens. Aminoguanidine is considered to react with the carbonyl group of Amadori or glycoxidation products and prevents the diabetic complications (29). If L-ARG has a biological activity similar to aminoguanidine, L-ARG may prevent NO quenching by advanced glycation endoproducts and augment the effect of ACh. Further studies are warranted.
Responses of vascular tonus
L-NAME significantly decreased basal CBF and significantly increased coronary vascular resistance in a dose-dependent manner in both vehicle-treated and diabetic dogs. Previous reports suggest that basal release of EDNO plays a role in the regulation of the CBF (4,5,17). Whether basal release of EDNO was impaired in the present diabetic model is not clear. However, we showed that L-ARG significantly increased CBF and significantly decreased CVR in diabetic dogs, but not in vehicle-treated dogs. We previously reported that the decrease in basal CBF after 10-5_M_ L-NAME was reversed by L-ARG (5). Therefore, the increase in basal CBF and the decrease in CVR after L-ARG in diabetic dogs suggest that L-ARG is a rate-limiting factor for the generation of NO in the coronary resistance vessels in such dogs.
In the present study, mean BP was significantly increased after 10-3_M_ of L-NAME in diabetic dogs, but it was not in vehicle-treated dogs. Our previous study showed that in nondiabetic dogs mean BP was significantly increased after 10-2_M_ of L-NAME (5). These two observations suggest that L-ARG becomes a rate-limiting factor for NO synthesis in the peripheral vessels of diabetic dogs and therefore that NO synthase is readily inhibited by a low dose of L-NAME in diabetic dogs.
Implication
Our results confirm previous observations in animal and humans of impairment of endothelium-dependent vasodilation in diabetes mellitus. EDNO not only causes vasodilation, but also has antiatherogenic properties (30). In the diabetic state, platelet aggregation, atherosclerosis, and microangiopathy are all enhanced. The impairment of EDNO may contribute to these pathologic conditions. In the present study, L-ARG restored ACh-induced flow increment and increased basal CBF. Therefore, its administration may prevent diabetic complications by potentiating EDNO.
Acknowledgment: This work was supported in part by Grant-in-Aid No. C02670401 for Scientific Research from the Ministry of Education and by a Research Grant from the Smoking Research Foundation, Tokyo, Japan.
The percent changes in coronary blood flow and coronary vascular resistance after acetylcholine (10, 30, 100 ng/kg) in vehicle-treated dogs (vehicle, open circles) and dogs with alloxan-induced diabetes (diabetes, solid circles). *p < 0.01 vehicle-treated dogs as compared with diabetic dogs.
The percent changes in coronary blood flow and coronary vascular resistance after adenosine (1 μg/kg) in vehicle-treated dogs (vehicle) and dogs alloxan-induced diabetes (diabetes).
Effects of intracoronary N G-nitro-L-arginine methyl (L-NAME) ester infusion (10-5-10-3 M in artery blood) on the percent changes in coronary blood flow (CBF) and coronary vascular resistance (CVR) after acetylcholine (right) or adenosine (left) in vehicle-treated dogs (vehicle, open circles) and alloxan-induced diabetic dogs (diabetes, solid circles). *p < 0.05 vehicle-treated dogs as compared with diabetic dogs.
Effects of L-arginine (L-ARG 150 mg/kg) on the percent changes in coronary vascular resistance after acetylcholine (10, 30, 100 ng/kg) or adenosine (1 μg/kg) in vehicle-treated dogs. Before L-ARG (open columns); after L-ARG (solid columns).
Effects of L-arginine, D-arginine, and Cu, Zn-superoxide dismutase (SOD) on the percent changes in coronary vascular resistance after acetylcholine (10, 30, 100 ng/kg) or adenosine (1 μg/kg) in dogs with alloxan-induced diabetes. *p < 0.05 and **p < 0.01 before (open columns) versus after (solid columns) treatment.
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Keywords:
Diabetes mellitus; Endothelium-derived nitric oxide; _N_G-Nitro-L-arginine methyl ester; L-arginine
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