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J Appl Physiol 87: 574-581, 1999;
8750-7587/99 $5.00
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Vol. 87, Issue 2, 574-581, August 1999

Interactions between angiotensin II and nitric oxide during exercise in normal and heart failure rats

J. David Symons1, Charles L. Stebbins1, and Timothy I. Musch2

1 Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, California 95616; and 2 Departments of Kinesiology and of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that nitric oxide (NO) opposes ANG II-induced increases in arterial pressure and reductions in renal, splanchnic, and skeletal muscle vascular conductance during dynamic exercise in normal and heart failure rats. Regional blood flow and vascular conductance were measured during treadmill running before (unblocked exercise) and after 1) ANG II AT1-receptor blockade (losartan, 20 mg/kg ia), 2) NO synthase (NOS) inhibition [NG-nitro-L-arginine methyl ester (L-NAME); 10 mg/kg ia], or 3) ANG II AT1-receptor blockade + NOS inhibition (combined blockade). Renal conductance during unblocked exercise (4.79 ± 0.31 ml · 100 g-1 · min-1 · mmHg-1) was increased after ANG II AT1-receptor blockade (6.53 ± 0.51 ml · 100 g-1 · min-1 · mmHg-1) and decreased by NOS inhibition (2.12 ± 0.20 ml · 100 g-1 · min-1 · mmHg-1) and combined inhibition (3.96 ± 0.57 ml · 100 g-1 · min-1 · mmHg-1;all P < 0.05 vs. unblocked). In heart failure rats, renal conductance during unblocked exercise (5.50 ± 0.66 ml · 100 g-1 · min-1 · mmHg-1) was increased by ANG II AT1-receptor blockade (8.48 ± 0.83 ml · 100 g-1 · min-1 · mmHg-1) and decreased by NOS inhibition (2.68 ± 0.22 ml · 100 g-1 · min-1 · mmHg-1; both P < 0.05 vs. unblocked), but it was unaltered during combined inhibition (4.65 ± 0.51 ml · 100 g-1 · min-1 · mmHg-1). Because our findings during combined blockade could be predicted from the independent actions of NO and ANG II, no interaction was apparent between these two substances in control or heart failure animals. In skeletal muscle, L-NAME-induced reductions in conductance, compared with unblocked exercise (P < 0.05), were abolished during combined inhibition in heart failure but not in control rats. These observations suggest that ANG II causes vasoconstriction in skeletal muscle that is masked by NO-evoked dilation in animals with heart failure. Because reductions in vascular conductance between unblocked exercise and combined inhibition were less than would be predicted from the independent actions of NO and ANG II, an interaction exists between these two substances in heart failure rats. L-NAME-induced increases in arterial pressure during treadmill running were attenuated (P < 0.05) similarly in both groups by combined inhibition. These findings indicate that NO opposes ANG II-induced increases in arterial pressure and in renal and skeletal muscle resistance during dynamic exercise.

regional blood flow; vascular resistance; vascular conductance; NG-nitro-L-arginine methyl ester; losartan


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING DYNAMIC EXERCISE a redistribution of cardiac output occurs such that blood flow is decreased to the renal and splanchnic circulations and increased to the heart and active skeletal muscle (28). We demonstrated previously that ANG II and endothelium-derived nitric oxide (NO) contribute significantly to these circulatory adjustments. For example, ANG II AT1-receptor blockade reduced mean arterial pressure (MAP) and vascular resistance in the myocardial, renal, and splanchnic vasculature in treadmill-running miniswine (34, 39). Conversely, NO synthase (NOS) inhibition increased MAP and vascular resistance in the renal, splanchnic, and skeletal muscle circulations in dynamically exercising rats (15).

Although ANG II and NO can influence vascular regulation independently, a relationship may exist between these two substances. Specifically, stimulation of ANG II AT1 receptors enhances the activity of endothelial NOS (12, 29) and indexes of NO generation (8), whereas inhibition of NOS augments ANG II-induced renal vasoconstriction (3, 17, 27). Recently, we observed in conscious rats that acute ANG II infusion does not reduce blood flow or conductance to the stomach or skeletal muscle unless NOS inhibition is present (38).

Because dynamic exercise increases ANG II and NO (22, 34, 42), an interaction between these two vasoactive agents could contribute to changes in arterial pressure and regional blood flow that occur during exercise. Furthermore, this potential interaction could be altered in heart failure because the renin-angiotensin system is activated to a greater degree, and the production of NO may be reduced (20, 36). Thus we examined the hypotheses that NO opposes ANG II-induced increases in arterial pressure and reductions in renal, splanchnic, and skeletal muscle vascular conductance during dynamic exercise in control and heart failure rats.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Normal Rats

Female Wistar rats (n = 27) were maintained on a 12:12-h light-dark cycle in a temperature-controlled environment and received food and water ad libitum. For 2 wk before surgery, animals were familiarized with running on a motorized treadmill. All surgical and experimental procedures were approved by the Animal Use and Care Committee at Kansas State University.

Surgical instrumentation. Rats were anesthetized initially by using 5% halothane-95% oxygen and were maintained during surgery with a 2% halothane-98% oxygen mixture. While the animals were breathing spontaneously, one catheter (PE-10 connected to PE-50 tubing) was placed in the right carotid artery and advanced to the ascending aorta while a second catheter was inserted into the caudal (tail) artery. Both catheters were flushed with heparinized saline, tunneled subcutaneously to the dorsal aspect of the cervical region, and exteriorized through an incision in the skin (1, 15, 23-25, 38). When all incisions were closed, anesthesia was terminated, and each rat was given approx 90-120 min to recover before completing one of four protocols. This period of recovery was chosen on the basis of a previous study indicating that cardiac or circulatory dynamics, regional blood flow, arterial blood gases, and acid-base status are stable at least 1 h after termination of halothane anesthesia (9).

Measurement of hemodynamic variables. The catheter placed in the ascending aorta was used for measuring arterial blood pressures, assessing heart rate (HR), and injecting radioactive microspheres to determine regional blood flow (19, 38). When regional blood flow was determined, arterial pressures and HR were monitored and recorded (model TA 4000, Gould, Cleveland, OH) immediately before and after each microsphere injection. Because blood pressures were similar before and after injection of microspheres for each protocol, the results were averaged. Regional blood flow was determined by injecting approx 0.5-0.6 × 106 microspheres (15 µm in diameter; New England Nuclear, Boston, MA) into the ascending aorta via the carotid artery catheter approx 15 s after withdrawal of the reference sample was initiated (0.250 ml/min; model 907, Harvard, Cambridge, MA) from the caudal artery (19, 38). Withdrawal was maintained for approx 90 s. Radiolabels were used in random order and included two of either scandium-46, cerium-141, tin-113, or strontium-85. Tissue and reference samples were analyzed for the quantity of gamma radiation from each isotope (model 5230, Packard Auto Gamma Spectrometer, Downers Grove, IL). Regional blood flow was calculated and expressed in milliliters per minute per 100 g of tissue (ml · min-1 · 100 g-1; Ref. 13). Adequate mixing of the microspheres was verified when blood flow differences between the right and left kidney and between the right and left hindlimb musculature were <15%. Regional vascular conductance (ml · 100 g-1 · min-1 · mmHg-1) was calculated by dividing each regional flow by MAP.

Protocol 1: Hemodynamic responses to exercise during NOS inhibition. This protocol assessed the contribution of NO to hemodynamic responses to dynamic exercise (n = 7; wt = 286 ± 6 g). After preexercise HR and blood pressure were recorded, rats began running up a 10% grade while the treadmill speed was increased progressively over approx 30 s to 20 m/min. Immediately after the measurement of HR and arterial pressure at approx 5 min of exercise, radioactive microspheres were injected into the ascending aorta via the carotid artery catheter to assess regional blood flow (19, 38).

After a rest period of 60-90 min, NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg ia) was infused over 1-2 min while arterial pressure was monitored. Five minutes after the infusion of L-NAME was completed, animals performed a second exercise bout at a speed and grade identical to those of the first bout. Radioactive microspheres were injected at 5 min of exercise to determine regional blood flow. The dose of L-NAME was chosen on the basis of preliminary studies indicating its ability to attenuate ACh-induced reductions in blood pressure in conscious rats (38).

Protocol 2: Hemodynamic responses to exercise during ANG II AT1-receptor inhibition. In this protocol, we evaluated the role of ANG II in the hemodynamic response to dynamic exercise (n = 8; wt = 359 ± 18 g). Hemodynamic variables were measured before and during the first exercise session as described in protocol 1. Ten minutes before the second exercise bout, a selective ANG II AT1-receptor antagonist [losartan; 20 mg/kg ia (32)] was administered, and preexercise and exercise hemodynamic variables were measured. Our dose of losartan was chosen on the basis of previous data using conscious miniswine (34, 39) and rats (38).

Protocol 3: Hemodynamic responses to exercise during ANG II AT1 receptor + NOS inhibition. This protocol assessed the effect of combined ANG II AT1-receptor + NOS inhibition on the hemodynamic responses to exercise (n = 8; wt = 307 ± 8 g). Preexercise and exercise hemodynamic variables were measured as described in protocol 1. Ten minutes before the second treadmill run, losartan (20 mg/kg ia) and (5 min later) L-NAME (10 mg/kg ia) were administered, and preexercise and exercise HR and blood pressure were recorded.

Protocol 4: Hemodynamic responses to exercise during vehicle administration. This protocol determined whether the inactive enantiomer of L-NAME [i.e., NG-nitro-D-arginine methyl ester (D-NAME)], the vehicle for losartan and L-NAME (i.e., saline), and/or the time period between exercise sessions (i.e., 60-90 min) altered any of the measured hemodynamic variables during treadmill running (n = 4; 331 ± 19 g). Two identical exercise bouts were performed. D-NAME (10 mg/kg ia) was administered over 1-2 min, 5 min before the second session.

Heart Failure Rats

Female Wistar rats (n = 35) were anesthetized as described in Surgical instrumentation, intubated, and ventilated mechanically (model 680, Harvard). After a left thoracotomy, the pericardium was opened and the heart was exposed. A suture (6-O Ethilon) then was passed under the left main coronary artery, and the vessel was ligated (n = 22). The resultant myocardial ischemia is sufficient to cause infarction that leads eventually to heart failure in 6-8 wk (1, 16, 23-25). In 13 sham-operated animals, the suture around the left main coronary artery was not ligated. After surgery, all animals were cage confined for 6-8 wk. Two weeks before the final experimental protocols, each rat was familiarized with treadmill running.

Surgical instrumentation. The purpose of this surgery was to measure left ventricular end-diastolic pressure (LVEDP) and to instrument the rats so that hemodynamic variables could be assessed before and during treadmill running. After the animals were anesthetized, a high-fidelity catheter (2F, Millar instruments, Houston, TX) was placed in the carotid artery and advanced retrogradely into the left ventricle (LV) to measure LVEDP. Next, the catheter was removed, and fluid-filled carotid and caudal artery catheters were inserted as described in this section. Approximately 90-120 min after termination of anesthesia (9), animals completed one of the following protocols.

Protocol 5: Hemodynamic responses to exercise during NOS inhibition. This protocol assessed the role of NO in the hemodynamic responses to dynamic exercise in heart failure (n = 7; wt = 255 ± 12 g) and sham-operated animals (n = 6; 301 ± 8 g). Procedures were identical to those described for protocol 1 in the normal rats.

Protocol 6: Hemodynamic responses to exercise during ANG II AT1-receptor inhibition and combined ANG II AT1-receptor + NOS inhibition. This protocol assessed the contribution of ANG II to the hemodynamic responses to treadmill running before and after the inhibition of NO-mediated vasodilation in heart failure (n = 11; wt = 256 ± 7 g) and sham-operated rats (n = 7; 287 ± 12 g). Ten minutes after losartan was given (20 mg/kg ia), preexercise and exercise hemodynamic variables were measured. Sixty to ninety minutes later, L-NAME (10 mg/kg ia) was injected. The second exercise session then was performed after 5 min.

Protocol 7: Reproducibility of the hemodynamic responses to treadmill running. This protocol was identical to protocol 4 (n = 5; 264 ± 8 g).

Verification of NOS Inhibition and ANG II AT1-Receptor Blockade

In protocols involving NOS inhibition (i.e., protocols 1, 3, 5, and 6), blood pressure was measured before and during a bolus injection of ACh (10 µg/kg ia) to demonstrate endothelium-dependent vasodilation in the absence of L-NAME. Approximately 10 min after completion of the second exercise bout (i.e., in the presence of L-NAME), the blood pressure response to ACh again was measured to quantify the degree of NOS inhibition. In the time/vehicle protocols (i.e., protocols 4 and 7), the endothelium-dependent response to ACh was recorded before the first and after the second bout of exercise to demonstrate reproducibility of ACh-induced blood pressure reductions.

In those protocols involving ANG II AT1-receptor blockade (i.e., protocols 2, 3, and 6) or reproducibility (i.e., protocols 4 and 7), the blood pressure response to a bolus injection of ANG II (1 µg/kg ia) was monitored ~10 min after the second exercise bout.

Postmortem Procedures

After the animals were euthanized (pentobarbital sodium, >50 mg/kg ia), the thorax was opened and placement of the carotid artery catheter into the aortic arch was verified. Next, the heart was excised, and the atria and ventricles were separated and weighed. Finally, the renal and splanchnic organs (stomach, small and large intestine) and skeletal muscle from the entire left hindlimb (measured as 10 pieces of approximately equal weight) were identified, removed, blotted, weighed, and placed in counting vials.

ANG II (Bachem, Torrance, CA), ACh, L-NAME, and D-NAME (Sigma Chemical, St. Louis, MO) were purchased commercially. Losartan was donated by Merck Research Laboratories (Wilmington, DE). All substances were reconstituted by using saline and made fresh daily from stock solutions.

Statistical Analysis

Data are expressed as group means ± SE. Nonparametric statistics were used because an initial analysis indicated that not all data were distributed normally. In the normal rats, MAP, HR, and regional blood flow responses were similar during exercise in the unblocked condition for protocols 1, 2, and 3. Therefore, the data were combined. Because the four resulting means from the normal rats (i.e., unblocked, NOS inhibition, ANG II AT1-receptor inhibition, and NOS + ANG II AT1-receptor inhibition) were similar to those obtained from the sham-operated animals, these data were combined, designated as control rats, and the means were compared by using the Kruskal-Wallis test statistic (11). This test also was used to compare the four means from the heart failure rats. The Wilcoxon signed-rank test was used when two means were compared (11).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Control Rats

Hemodynamic responses to NOS inhibition. Relative to the unblocked condition, NOS inhibition increased MAP and decreased HR both before and during treadmill running (Table 1). In the renal, splanchnic, and skeletal muscle circulations, blood flow and vascular conductance were reduced during exercise + L-NAME compared with the unblocked condition (Table 2).

                              
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  		Table 1.   Mean arterial pressure and heart rate in control rats before and during exercise


                              
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  		Table 2.   Splanchnic, renal, and skeletal muscle blood flow and vascular conductance in control rats during exercise

ACh-induced reductions in MAP before and after administration of L-NAME were 37 ± 2 and 24 ± 2% (P < 0.05), respectively.

Hemodynamic responses to ANG II AT1-receptor inhibition. At rest, MAP and HR after ANG II AT1-receptor inhibition were similar to unblocked conditions. Compared with NOS inhibition, however, MAP was lower and HR was higher after losartan. During exercise, MAP was lower after losartan compared with the unblocked condition. MAP was lower and HR was higher during exercise after losartan compared with L-NAME (Table 1).

After ANG II AT1-receptor inhibition, renal flow and conductance were greater compared with the unblocked condition but were similar in the skeletal muscle and splanchnic regions during exercise. Compared with L-NAME, blood flow and conductance were higher in every circulation after losartan (Table 2). Exogenous ANG II increased MAP by 6 ± 3% (P < 0.05 vs. ANG II administration after vehicle, i.e., protocol 4).

Hemodynamic responses to ANG II AT1-receptor + NOS inhibition. NOS + ANG II AT1-receptor inhibition increased MAP and reduced HR before and during exercise compared with both the unblocked and ANG II AT1-receptor inhibition conditions. In contrast, at rest and during treadmill running, MAP was lower and HR was similar after combined inhibition compared with L-NAME alone (Table 1).

Compared with exercise in the unblocked condition, blood flow and conductance were reduced in all vascular beds. An exception to this occurred in the skeletal muscle where vascular conductance was lower after combined compared with unblocked exercise, but blood flow was similar. In contrast, blood flow and/or conductance was higher in the renal circulation, but lower in the large intestine, after combined compared with NOS inhibition. Lower values for flow and/or conductance were observed during combined blockade than during ANG II AT1-receptor inhibition (Table 2).

ACh-induced reductions in MAP before and after L-NAME were 42 ± 2 and 24 ± 3% (P < 0.05), respectively. Exogenous ANG II increased MAP 4 ± 1% (P < 0.05 vs. ANG II administration after vehicle, i.e., protocol 4).

Absolute changes in vascular conductance from unblocked exercise in the renal and skeletal muscle circulations in response to L-NAME, losartan, and combined inhibition are shown in Fig. 1A. Also presented are changes from unblocked exercise to combined inhibition that would be expected to occur if the effects of losartan and L-NAME simply were additive.


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  		Fig. 1.   Change from unblocked exercise in skeletal muscle and renal vascular conductance after nitric oxide synthase inhibition [NG-nitro-L-arginine methyl ester (L-NAME)], ANG II AT1-receptor blockade (losartan), or combined inhibition (L-NAME + losartan) in control (A) and heart failure (B) rats. Predicted, L-NAME-induced reductions in conductance + losartan-induced increases in conductance. Error bars are not shown for predicted values. Values are means ± SE. * P < 0.05 vs. unblocked exercise. dagger  P < 0.05 vs. L-NAME. ddager  P < 0.05 vs. control rats.

Hemodynamic responses to administration of vehicle. D-NAME had no effect on MAP, HR, or regional blood flow and conductance at rest or during exercise. For brevity, these data are not shown.

ACh-induced reductions in MAP were 43 ± 7 and 48 ± 10% before and after D-NAME, respectively. Exogenous ANG II increased MAP 23 ± 3%. This increase was greater (P < 0.05) than that observed after the second treadmill in the presence of losartan (protocol 2) or losartan + L-NAME (protocol 3).

Heart Failure Rats

LVEDP and the right ventricle (RV) weight-to-body weight ratio were similar among the heart failure animals used for protocols 5, 6, and 7. Consequently, the data were combined. LVEDP was 16 ± 1 mmHg and the RV weight-to-body weight ratio was 0.68 ± 0.05 mg/g (n = 22). In the sham-operated animals (n = 13), LVEDP (7 ± 1 mmHg) and the RV weight-to-body weight ratio (0.57 ± 0.03 mg/g) were lower (P < 0.05) compared with those of heart failure rats.

Hemodynamic responses to NOS inhibition. Compared with the unblocked condition, MAP was greater, whereas HR was less, at rest and during exercise after L-NAME (Table 3). During exercise, NOS inhibition reduced regional blood flow and conductance to all circulations (Table 4). ACh-induced reductions in MAP were 24 ± 3 and 14 ± 5% (P < 0.05) before and after L-NAME, respectively.

                              
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  		Table 3.   Mean arterial pressure and heart rate in heart failure rats before and during exercise


                              
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  		Table 4.   Splanchnic, renal, and skeletal muscle blood flow and vascular conductance in heart failure rats during exercise

Hemodynamic responses to ANG II AT1-receptor inhibition. Before exercise, HR was greater after ANG II AT1-receptor inhibition compared with the unblocked and L-NAME conditions (Table 3). During exercise, MAP was similar to unblocked conditions but was lower than after NOS inhibition.

Of the regional circulations evaluated, only renal blood flow and conductance were greater during treadmill running after ANG II AT1-receptor inhibition than in the unblocked condition. Compared with NOS inhibition, flow and conductance were greater in all of the circulations after losartan (Table 4).

Hemodynamic responses to ANG II AT1-receptor + NOS inhibition. MAP was higher and HR lower after combined inhibition compared with ANG II AT1-receptor blockade both before and during treadmill running (Table 3).

Compared with unblocked conditions, blood flow and conductance after combined inhibition were similar in the renal and skeletal muscle circulations but were lower in the splanchnic region. Perfusion to the renal and skeletal muscle circulations was greater after combined blockade compared with NOS inhibition alone. Both renal and splanchnic flow and conductance were reduced after L-NAME + losartan compared with losartan alone (Table 4).

ACh-induced reductions in MAP were 28 ± 4 and 19 ± 4% after losartan and losartan + L-NAME, respectively (P < 0.05). Exogenous ANG II increased MAP by 9 ± 2% (P < 0.05 vs. ANG II administration after vehicle, i.e., protocol 7).

Changes in absolute renal and skeletal muscle vascular conductance from unblocked exercise in response to L-NAME, losartan, and combined inhibition are shown in Fig. 1B. In addition, the values that would be predicted to occur during combined blockade if the effects of losartan and L-NAME were additive also are presented.

Reproducibility of the hemodynamic responses to treadmill running. The vehicle for losartan and L-NAME had no effect on MAP, HR, or regional blood flow and conductance at rest or during exercise. For brevity, these data are not shown. Exogenous ANG II increased MAP 28 ± 3%. This response was greater (P < 0.05) than that observed after losartan + L-NAME (i.e., protocol 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Results from this study support our hypothesis that NO opposes the effects of ANG II on arterial pressure and regional blood flow. Specifically, NO antagonized ANG II-evoked increases in blood pressure and decreases in renal conductance in both heart failure and control rats. In skeletal muscle, ANG II caused vasoconstriction in the heart failure rats that could only be seen during NOS inhibition. Finally, ANG II did not alter conductance in the splanchnic vasculature of heart failure or control animals or the skeletal muscle of control rats.

ANG II AT1-Receptor Inhibition During Exercise

In normal rats, inhibition of ANG II AT1 receptors elevated renal blood flow and conductance and reduced arterial pressure during exercise. Our finding that splanchnic conductance was similar after losartan compared with the unblocked condition was unexpected because losartan can reduce arterial pressure and increase myocardial, renal, and splanchnic blood flow in treadmill-running miniswine (34, 39). This discrepancy probably is due to the fact that our rats ran for ~5 min at ~50% of their maximal oxygen consumption (VO2 max), whereas the miniswine exercised for ~20 min at ~80% VO2 max. Therefore, there likely was less activation of the renin-angiotensin system in the exercising rats than in the pigs. The lower intensity and duration of exercise used in the present study were necessary because the willingness of the rats to run was reduced by NOS inhibition (see NOS Inhibition During Exercise). In any case, activation of the renin-angiotensin system can still occur during short-term (e.g., 10 min), low-intensity (e.g., 30% VO2 max) dynamic exercise (42). Evidence of such activation in our exercise protocol was that ANG II AT1-receptor blockade elevated renal blood flow and conductance and lowered arterial blood pressure compared with the unblocked condition. In the heart failure rats, analogous blockade increased renal flow and conductance by >50%. However, no effects were observed in other regional circulations or on arterial pressure. Relative to unblocked conditions, ANG II AT1-receptor blockade probably caused larger elevations in renal conductance in heart failure compared with control rats (54 vs. 36%, respectively) because the renin-angiotensin system was activated to a greater degree (18, 31, 37). The finding that losartan did not alter arterial pressure during exercise in the heart failure animals was unexpected. It may be that MAP was maintained, in part, by an increased cardiac output, because HR was elevated significantly during exercise + losartan.

NOS Inhibition During Exercise

In both groups of rats, NOS inhibition increased arterial pressure and reduced blood flow and conductance in the renal, splanchnic, and skeletal muscle circulations during exercise. These observations are consistent with those reported previously (15, 16), even though we used a lower dose of L-NAME (i.e., 10 vs. 30-100 mg/kg). The lower concentration was chosen because some rats were unwilling to run when higher doses were given [i.e., 30-100 mg/kg (15); pilot data (41)]. Preliminary studies also revealed that the length of time between administration of L-NAME and the initiation and completion of exercise was important. For example, some animals would not run even after 10 mg/kg if >10 min elapsed between L-NAME administration and the initiation of exercise and/or if the intensity of exercise was >20 m/min. Because of these limitations, exercise was initiated 5 min after administration of L-NAME at a work intensity of 20 m/min. This workload was sufficient to test our hypothesis because arterial pressure, skeletal muscle blood flow, and splanchnic and renal vascular resistance all increased from baseline values (38). The 10 mg/kg dose of L-NAME was effective because ACh-induced reductions in blood pressure were similar (i.e., ~24%) to those observed after 30 mg/kg L-NAME [i.e., ~26%; (15)].

ANG II AT1-Receptor + NOS Inhibition During Exercise

Our most interesting observations occurred during combined blockade. Compared with unblocked conditions, L-NAME-induced reductions in renal conductance in both groups of animals were practically eliminated by combined inhibition (Fig. 1). However, these effects appeared to be primarily additive. In other words, the outcome was predictable on the basis of the expected sum of independent L-NAME-induced decreases and losartan-evoked increases in renal conductance (Fig. 1). Thus, although the vasoactive effects of NO and ANG II offset each other, there appeared to be no interaction between them.

In skeletal muscle, our results support an interaction between ANG II and NO in the heart failure but not in the control rats. Although losartan did not increase skeletal muscle conductance in either group, decreases in conductance caused by L-NAME in the heart failure rats were abolished during combined blockade. If the actions of L-NAME and losartan were additive in this situation, then no change in L-NAME-induced vasoconstriction should have occurred (Fig. 1). These findings indicate that ANG II did not affect the skeletal muscle vasculature of normal rats during exercise. In the heart failure animals, however, this hormone caused vasoconstriction that was masked by vasodilation evoked by NO. Although the explanation for this interaction is not clear, skeletal muscle constriction may result from higher circulating levels of ANG II that are known to occur in heart failure (18, 20, 37).

The splanchnic vasculature of both groups of animals responded differently to combined inhibition than did the skeletal muscle (of heart failure animals) or renal circulations because losartan did not affect L-NAME-induced reductions in splanchnic flow or conductance. Thus decreases in splanchnic conductance (and skeletal muscle conductance of control rats) during moderate-intensity exercise after NOS inhibition were due to removal of NO-mediated opposition to vasoconstrictors other than ANG II [e.g., catecholamines (28, 42) other vasoconstrictor hormones (33-35, 40); general sympathetic neural activation (28)].

Higher arterial pressures during exercise and NOS inhibition likely were due to simultaneous reductions in conductance among regional circulations compared with the unblocked condition. Relative to exercise + L-NAME, lower systemic pressures during combined blockade probably were caused by the ability of losartan to offset ANG II-induced renal and skeletal muscle constriction.

Results from the heart failure rats may have important clinical implications. As mentioned, heart failure is associated with elevated plasma concentrations of ANG II (18, 31, 37). In addition, the vasodilator capacity of the peripheral circulation also may be impaired (7, 8). Consequently, the ability of NO to oppose ANG II-induced constriction may be compromised. Our results indicate that, in rats with heart failure, NO antagonizes ANG II-induced vasoconstriction in the renal and skeletal muscle circulations during exercise and that these effects are capable of reducing systemic pressure. Therefore, interventions that enhance the effects of NO, and/or reduce the action of ANG II, may lower the metabolic demand on the heart and perhaps increase exercise capacity.

Data from the present study are strengthened by three factors. First, the hemodynamic responses to two treadmill bouts were not affected by the time between exercise sessions and/or by administration of vehicle or volume in control and heart failure rats. Second, regional conductance was calculated and used to account for changes in regional vasomotor responses that occur in response to arterial pressure alterations (26). Third, the ability of the antagonists (e.g., losartan, L-NAME) to attenuate blood pressure responses to their respective agonists (e.g., ANG II, ACh) was documented in every animal.

Systemic administration of losartan and/or L-NAME may have caused central nervous system (CNS) effects that contributed to our findings. For example, ANG II can diminish baroreflex control of HR by resetting the operating point of the arterial baroreflex toward higher pressures (10, 21). Thus, during exercise + losartan, a lower HR would have been expected in our animals. However, this was not observed in either the control or heart failure rats. Consequently, it does not appear that ANG II exerted its major action in the CNS.

NO also can act in the CNS to affect sympathetic nerve activity that could alter regional blood flow and/or conductance. For instance, in anesthetized rats, NOS inhibition using NG-monomethyl-L-arginine or L-NAME can increase or decrease sympathetic nerve activity, respectively (14, 30). On the other hand, in the conscious state, L-NAME-induced regional or renal vasoconstriction is not altered by respective sympathetic nerve block in sheep (via thoracic epidural nerve block) (4) or renal denervation in rats (2). Thus the peripheral effects of L-NAME appear to predominate over the CNS effects in conscious animals.

Anesthesia and/or surgery may have caused increases in ANG II that influenced our results. Although this is a possibility that we cannot dismiss, it is unlikely because the half-life of ANG II is 2-4 min, and the time between surgery/anesthesia and data collection was ~90 min. Moreover, if ANG II levels were elevated ~90 min after surgery, then arterial pressure probably would be lower, and regional blood flow higher, when measured 24 h later. However, preliminary data from five rats indicate that arterial pressure (122 ± 7 and 122 ± 6 mmHg) and renal blood flow (680 ± 39 and 661 ± 65 mg · 100 g-1 · min-1) are similar when measured ~90 min or 24 h postsurgery.

The extent of LV infarction was not assessed in our heart failure rats. However, we believe our animals were in a state of moderate LV dysfunction. Delp et al. (5) used procedures identical to our own and reported that heart failure rats (LVEDP = 11 mmHg; RV weight-to-body weight ratio = 0.71 mg/g) had infarctions comprising 45% of the endocardial circumference, whereas sham-operated animals (LVEDP = 5 mmHg; RV weight-to-body weight ratio = 0.53 mg/g) had no discernable LV necrosis. These investigators classified their infarcted rats as having moderate LV dysfunction. Because LVEDP and RV weight-to-body weight ratio in their sham-operated and heart failure rats were similar to those of our rats (see RESULTS), we conclude that our heart failure rats also had moderate LV dysfunction.

Skeletal muscle blood flow during exercise can be compromised in rats with heart failure (16, 25). This phenomenon was not observed in the present investigation. However, reductions in hindlimb flow apparently depend on the degree of LV dysfunction (16). For instance, hindquarter perfusion during exercise was similar between sham-operated rats and those with infarcts comprising ~25-44% of the LV but was diminished in animals with infarctions >44% (16). Therefore, it seems that skeletal muscle blood flow during exercise in our heart failure rats was not impaired in the unblocked condition because LV dysfunction was moderate.

In summary, we found that NO-evoked dilation limited ANG II-induced renal vasoconstriction during exercise to a greater extent in heart failure than in normal rats. However, this response was due to an additive effect of the independent actions of these two substances. In skeletal muscle, NO masked ANG II-induced vasoconstriction in heart failure but not in control animals. ANG II had no effect on the splanchnic circulation in either group or the skeletal muscle of control animals. These findings suggest that the constrictive effects of ANG II during moderate-intensity dynamic exercise are enhanced in heart failure associated with moderate LV dysfunction.


    ACKNOWLEDGEMENTS

The authors acknowledge the cooperation of Drs. Jon Dunn, Michael Kenney, Chris Ross, and David Poole throughout this study. Technical assistance from K. Sue Hageman was greatly appreciated.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants AG-11535 (T. I. Musch) and HL-48372 (C. L. Stebbins); American Heart Association, Western States Affiliate, Grant-in-Aid 98-201; a Losartan Medical School Grant from Merck Research Laboratories; and research funds provided by the Division of Cardiovascular Medicine at the University of California Davis (J. D. Symons). These studies were performed while J. D. Symons was a Visiting Scientist at Kansas State University.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. D. Symons, Univ. of California, Davis, Dept. of Internal Medicine, Div. of Cardiovascular Medicine, TB 172, Davis, CA 95616 (E-mail: jdsymons{at}ucdavis.edu).

Received 11 September 1998; accepted in final form 29 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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