Angiotensin II–Nitric Oxide Interaction on Sympathetic Outflow in Conscious Rabbits
Abstract—Increasing evidence suggests that endogenous NO inhibits sympathetic outflow in anesthetized animals. However, in a recent study from this laboratory, we were unable to find any evidence of increased renal sympathetic nerve activity (RSNA) in response to blockade of NO synthesis in conscious rabbits. Because angiotensin II (Ang II) increases sympathetic outflow, one factor for this discrepancy may be the difference in the resting level of Ang II, which may be lower in well-trained conscious animals. In the present study, the effects of blockade of NO synthesis with Nω-nitro-l-arginine methyl ester (L-NAME, 30 mg/kg IV) on resting RSNA with and without a background intravenous infusion of Ang II (10 ng · kg−1 · min−1) was investigated in conscious rabbits. Intravenous administration of L-NAME (30 mg/kg) caused an increase in mean arterial blood pressure (MAP, from 80.4±2.9 to 92.8±2.5; P=.0001) and a decrease in RSNA (from 100±0% to 53.4±8.6%, P=.0016). When the elevated blood pressure was returned to control by infusion of hydralazine (0.01 to 0.06 mg · kg−1 · min−1), RSNA returned to the level before L-NAME administration. During a sustained infusion of Ang II (10 ng · kg−1 · min−1), L-NAME increased MAP from 89.2±2.9 to 109.0±4.3 mm Hg (P=.0101) and decreased RSNA from 100.0±0% to 53.7±7.5% (P=.0013). Under this circumstance, however, when the MAP was returned to the level that existed before the administration of L-NAME, RSNA increased significantly above the level that existed before the administration of L-NAME (164.5±17.7% versus 100±0%, P=.0151). The enhancement of the sympathetic response by Ang II was completely blocked by the AT1 receptor antagonist, losartan. In contrast, during a background infusion of phenylephrine, which increased MAP to the same level as produced by Ang II, L-NAME had no effect on RSNA when MAP was returned to the control level. Nω-Nitro-d-arginine methyl ester had no effect on MAP and RSNA. Intravenous infusion of Ang II alone for 75 minutes had no effect on RSNA when MAP was returned to control levels. These data suggest that an elevated level of Ang II is critical for the inhibitory effect of NO on sympathetic outflow in conscious rabbits and imply that these two substances have a major impact on the regulation of sympathetic outflow.
Several studies have recently reported a sympathoexcitatory effect after the administration of several analogues of the amino acid l-arginine, such as L-NAME and L-NMMA.1 2 This effect ostensibly contributes to the hypertensive response after blockade of NO synthesis.1 3 Augmentation of sympathetic function has been observed by direct recording of RSNA2 and lumbar1 sympathetic nerve activity in anesthetized rats and cats. It has also been shown that inhibition of NO synthase enhances the action of norepinephrine postsynaptically.4 The major sympathoexcitatory effect of inhibition of NO synthase stems from its action on several midbrain nuclei, including the NTS,5 the paraventricular nucleus,6 and the RVLM.7 However, in a recent study from this laboratory,8 we did not find any sympathoexcitatory effect of inhibition of NO synthase on resting RSNA even in the chronic sinoaortic baroreceptor–denervated state. This observation confirmed a recent study by Hansen et al.9 These investigators failed to show an excitatory effect of L-NMMA on muscle sympathetic nerve activity in normal resting human subjects.
Interactions between Ang II and NO have been observed in a variety of tissues,10 11 12 13 including the central nervous system.14 Bains and Ferguson15 have demonstrated that inhibition of NO synthase enhanced the pressor response to electrical stimulation of the subfornical organ. This area is known to be rich in Ang II and uses Ang II as a neurotransmitter.14 16 The interaction between Ang II and NO has also been shown in the microcirculation. For instance, Haberl et al17 showed a delayed cerebral vasodilation after pial application of Ang II that was significantly attenuated by inhibition of NO synthase.
The majority of studies have demonstrated a sympathoexcitation in response to inhibition of NO synthase in anesthetized preparations and have not been reproduced in conscious animals8 or humans.9 In regard to the known interactions between NO and Ang II in the peripheral10 11 13 and central nervous systems,12 14 we hypothesized that elevated levels of Ang II that may be present under anesthesia18 19 might explain the discrepancy concerning the sympathoexcitation induced by blockade of NO synthesis. Accordingly, we evaluated the effects of blocking NO synthesis on RSNA in conscious rabbits with and without a background infusion of a low dose of Ang II. These data show that an increase in circulating Ang II is critical for the sympathoexcitation induced by NO synthase inhibition.
Materials and Methods
Animals and Surgical Instrumentation
Studies were carried out on 30 male New Zealand White rabbits ranging in weight between 2.5 and 3.5 kg. All surgical procedures and protocols were reviewed and approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Experiments were carried out under the Guidelines for the Care and Use of Experimental Animals of the American Physiological Society and the National Institutes of Health. Rabbits were kept in individual cages in a temperature-controlled room (23°C) and fed a standard rabbit chow (Harlan Techlab) consisting of 0.29% Na+ and 1.4% K+.
For surgical instrumentation, rabbits were anesthetized with a cocktail consisting of 1.2 mg/kg acepromazine, 5.9 mg/kg xylazine, and 58.8 mg/kg ketamine given as an intramuscular injection. Supplemental anesthesia was provided by intravenous pentobarbital sodium at a dose of 1.7 mg/kg as needed. After tracheal intubation, sterile surgery was carried out to implant a renal sympathetic nerve electrode and arterial catheter as previously described.8 In brief, a left subcostal incision was made, and the kidney was approached in the retroperitoneal space. A bundle of renal nerves was identified and gently freed from surrounding tissue using glass rods. Two polytetrafluoroethylene-coated stainless-steel wire electrodes (outer diameter, 0.124 mm; A-M Systems) were placed around the dissected renal nerves. To insulate the electrodes and the nerve from the surrounding tissue and to prevent the nerves from drying, the electrodes and the nerve assembly were covered with a two-component silicone gel (Wacker Sil-Gel). A ground lead was sutured to the fat close to the electrodes. The electrodes and the ground lead were tunneled beneath the skin to the back and fixed between the shoulder blades. The flank incision was closed.
Through a midline cervical incision, a Micro-Renathane catheter (outer diameter, 0.065 in; inner diameter, 0.030 in; Braintree Scientific) was inserted into the left carotid artery for the measurement of arterial pressure and HR. The catheter was tunneled beneath the skin and brought out the back of the neck. The catheter was flushed daily with heparin sodium (1000 U/mL, Elkins-Sinn). After the surgery, the rabbits were treated for 3 days with enrofloxacin (2.3 mg/kg IM, twice per day; Baytril, Miles).
Hemodynamic and Sympathetic Nerve Recording
Arterial blood pressure was recorded with a Hewlett-Packard pressure transducer and a Gould bridge amplifier. HR and MAP were derived by the data acquisition software (MacLab) using the arterial pressure pulse. The renal sympathetic nerve electrode wires were attached to a Grass P16 preamplifier with the band-pass filters set between 100 Hz and 1 kHz. The amplified signal was displayed on a storage oscilloscope and passed through an audio amplifier and loudspeaker. The raw nerve activity was full wave–rectified and integrated using the MacLab software. In addition to integrated nerve activity, the frequency of discharge (in spikes per second) was recorded using the frequency function of MacLab. A window discriminator was set above the noise level in order to use the ratemeter function of the MacLab system. Background noise was determined when arterial pressure was increased with PE. The integrated noise level was subtracted from the integrated nerve activity.
Experiments were carried out 3 to 7 days after implantation of the renal nerve electrodes. Rabbits were trained to sit quietly in a Lucite box of our own design. On the day of the experiment, the arterial catheter was connected to a pressure transducer, and MAP and HR were measured. The renal sympathetic nerve electrode wires were attached to the preamplifier. Raw RSNA, integrated RSNA, and the frequency of RSNA were recorded. After the rabbit was attached to the recording equipment, it was allowed to rest for ≈30 minutes before any data were taken. To minimize the effect of the alterations in HR on RSNA,20 the rabbit was pretreated with atropine methylbromide (2.0 mg/kg IV) plus metoprolol (2.0 mg/kg IV) to block both sympathetic and parasympathetic influences on the heart; this pretreatment increased HR from 215±5.6 to 236±9.4 bpm (P=.0120). During the following protocols, HR did not change after the administration of L-NAME, Ang II, PE, or hydralazine.
Protocol 1: Responses to Intravenous L-NAME (n=7)
After a control period of 20 minutes, L-NAME (30 mg/kg) was infused at 6.0 mg · kg−1 · min−1. In order to minimize baroreflex activation, 15 minutes after completion of the infusion of L-NAME, MAP was brought back to control by intravenous infusion of hydralazine (0.01 to 0.06 mg · kg−1 · min−1). All hemodynamics and RSNA were recorded continuously.
Protocol 2: Responses to Intravenous L-NAME With a Background Intravenous Infusion of Ang II (n=7)
After a control period of 20 minutes, an intravenous background infusion of Ang II was begun at a rate of 10 ng · kg−1 · min−1. Sixty minutes after beginning the Ang II infusion, L-NAME was infused at 6.0 mg · kg−1 · min−1. Fifteen minutes after completion of the L-NAME infusion, MAP was returned to the level that existed before the administration of L-NAME by intravenous infusion of hydralazine. Ang II was continuously infused over this period. All hemodynamics and RSNA were recorded continuously.
Protocol 3: Responses to Intravenous L-NAME With a Background Intravenous Infusion of PE (n=5)
In order to differentiate between the effects of Ang II per se and its acute pressor effect, protocol 2 was repeated except that the Ang II infusion was substituted with a PE infusion. The dose of PE (1 to 5 μg · kg−1 · min−1) was titrated so as to match the increase in MAP produced by Ang II.
Protocol 4: Responses to Intravenous D-NAME With a Background Intravenous Infusion of Ang II (n=5)
To test the specificity of the effect of L-NAME, its inactive analogue, D-NAME (30 mg/kg), was substituted for L-NAME. The other procedures were the same as those described in protocol 2.
Protocol 5: Responses to Intravenous L-NAME With a Background Intravenous Infusion of Ang II After Blockade of the AT1 Receptor (n=5)
To determine whether the effect of Ang II is mediated by stimulation of the AT1 receptor, a specific AT1 receptor antagonist, losartan (5 mg/kg), was injected intravenously 15 minutes before infusion of Ang II. Blockade of AT1 receptors was demonstrated by the complete inhibition of the pressor response to a bolus injection of Ang II (0.1 μg). Protocol 2 was then repeated.
Protocol 6: Responses to Intravenous Ang II (n=6)
After a control period of 20 minutes, Ang II was infused intravenously at 10 ng · kg−1 · min−1. Seventy-five minutes after the beginning of the infusion of Ang II, hydralazine (0.01 to 0.06 mg · kg−1 · min−1) was infused simultaneously in order to return MAP to the control level. All hemodynamics and RSNA were recorded continuously.
L-NAME, D-NAME, Ang II, losartan, and atropine methyl bromide were obtained from Sigma Chemical Co. Metoprolol was obtained from CIBA Pharmaceutical. All drugs were made fresh on the day of the experiment.
Changes in RSNA were similar when quantified using either voltage integration or spike discharge rate. The discharge data in the present study are presented using the ratemeter method. All measurements were averaged every 30 seconds. In protocol 1, RSNA was expressed as the percentage of resting nerve activity that existed before drug administration. The resting nerve activity was set at 100%. Because background infusion of Ang II or PE increased MAP and decreased RSNA, in order to facilitate the comparison of the responses of RSNA induced by L-NAME under different conditions, in the other protocols (protocol 2 to 6), the RSNA level that existed just before L-NAME administration was renormalized to 100%. RSNA after L-NAME and hydralazine were expressed as the percentage of the nerve activity that existed before L-NAME administration. These data are presented as the mean±SEM. Multivariate analysis and repeated measures ANOVA procedures were used in conjunction with specific orthogonal contrasts for post hoc analysis. The P values reported are after adjustments using the Greenhouse-Geisser corrections for multisample sphericity. All statistical analyses were performed using the Statistical Analysis Systems (SAS) software. A value of P<.05 was considered statistically significant.
Effects of L-NAME Alone on MAP and RSNA
Fig 1⇓ is an original recording showing the responses to intravenous infusion of L-NAME in one rabbit. The mean data from this protocol are shown in Fig 2⇓. As can be seen from these two figures, intravenous administration of L-NAME in conscious, cardiac autonomic nervous system–blocked rabbits caused an increase in MAP (from 80.4±2.9 to 92.8±2.5 mm Hg, P=.0001) and a decrease in RSNA (from 100% to 53.4±8.6%, P=.0016). When the blood pressure was returned to control (79.9±2.5 versus 80.4±2.9 mm Hg, P=.5386) by infusing hydralazine, RSNA returned to near control levels (88.4±7.5% versus 100±0%, P=.1744).
In two additional rabbits, we also evaluated the effect of another NO synthase inhibitor, L-NNA, on RSNA. Similar results were observed for this analogue. When the increased MAP induced by L-NNA was returned to the control level, RSNA was also returned the control level (88.0±4.6% versus 100±0%).
Effects L-NAME on RSNA With a Background Intravenous Infusion of Ang II
Fig 3⇓ is a representative original recording from one rabbit showing the effects of L-NAME on MAP, HR, and RSNA with a background infusion of Ang II. Intravenous infusion of Ang II (10 ng · kg−1 · min−1) significantly increased MAP (P=.0138) and decreased RSNA (P=.0117). Fig 4⇓ shows the MAP and RSNA responses to L-NAME and hydralazine during a background infusion of Ang II. With the background infusion of Ang II, L-NAME increased MAP (from 89.5±3.0 to 109.0±4.3 mm Hg, P=.0101) and decreased RSNA (from 100±0% to 53.7±7.5%, P=.0013). When MAP was returned to the level before the administration of L-NAME (MAP, 89.7±2.2 versus 89.5±3.0 mm Hg [P=.6383]; pulse pressure, 28.2±2.3 versus 27.8±3.3 mm Hg [P=.8666]) by infusing hydralazine, RSNA increased to a level significantly higher than that before the administration of L-NAME (164.5±17.7% versus 100±0%, P=.0151).
In two additional animals, the effects of L-NNA on RSNA with a background infusion of Ang II were examined. When MAP was brought back to the level that existed before L-NNA administration, a definite overshoot of RSNA was observed (189.0±16.5% versus 100±0%).
Effects of L-NAME With a Background Intravenous Infusion of PE
Intravenous infusion of PE increased MAP (from 83.1±1.4 to 93.1±2.9 mm Hg, P=.0234) and decreased RSNA (from 100.0±0% to 69.8±0.8%, P=.0079) in a fashion similar to that for Ang II. As can be seen in Fig 5⇓, during a background infusion of PE, L-NAME increased MAP (from 93.1±2.9 to 112.9±3.8 mm Hg, P=.0101) and decreased RSNA (from 100±0% to 40.0±10.0%, P=.0001). When MAP was returned to the level that existed during the PE infusion before L-NAME (93.0±2.7 versus 93.1±2.9 mm Hg, P=.6383), RSNA was increased back to the level that existed before L-NAME infusion (98.0%±8.1% versus 100%±0%, P=.8537).
Effects of D-NAME With a Background Intravenous Infusion of Ang II
Fig 6⇓ shows the responses to the stereoisomer D-NAME during a background infusion of Ang II. Intravenous infusion of Ang II (10 ng · kg−1 · min−1) for 1 hour caused an increase of MAP (from 79.9±2.6 to 91.0±2.4 mm Hg, P=.0234) and a decrease in RSNA (from 100±0% to 50.5±10.2%, P=.0079). D-NAME had no further effects on MAP (91.0±2.1 versus 91.6±2.6 mm Hg, P=.8345) and RSNA (100±0% versus 99.9±2.8%, P=.6905).
Effects of L-NAME With a Background Intravenous Infusion of Ang II After Blockade of AT1 Receptors
Fig 7⇓ shows the mean data for this group. Intravenous injection of the AT1 receptor antagonist losartan (5 mg/kg) had no effect on resting MAP (81.8±2.7 versus 80.6±2.9 mm Hg, P=.2675) and RSNA (100±0% versus 98.5±8.4%, P=.2675). Ang II infusion had no effect on MAP (80.6±2.9 versus 80.9±1.6 mm Hg, P=.7997) and RSNA (98.5±8.4% versus 104.0±5.7%, P=.5595) after losartan. L-NAME increased MAP (from 80.9±1.6 to 93.2±2.0 mm Hg, P=.0005) and decreased RSNA (from 100±0% to 46.3±8.8%, P=.0037). When MAP was brought back to the level that existed before the infusion of L-NAME (80.7±1.9 versus 80.9±1.6 mm Hg, P=.7473), RSNA was not different from the level before the administration of L-NAME (91.1±4.4% versus 100±0%, P=.1129).
Effects of Ang II Infusion Alone on MAP and RSNA
Intravenous infusion of Ang II increased MAP from 82.7±2.2 to 92.1±2.4 mm Hg (P=.0205) and decreased RSNA from 100±0% to 62.9±5.8% (P=.0001). When MAP was returned to the control level (82.2±2.5 versus 82.7±2.2 mm Hg, P=.8930) by infusing hydralazine, RSNA was not different from control (103.7±1.9% versus 100±0%, P=.5112).
The new finding in the present study is that Ang II enhances the effect of blockade of NO synthesis on sympathetic nerve activity in conscious rabbits. During a background infusion of Ang II, L-NAME significantly enhanced RSNA when MAP was returned to the level that existed before the administration of L-NAME. These results suggest that elevated levels of central Ang II may be necessary in order to evoke an increase in sympathetic outflow after blockade of NO synthesis.
Previous studies conducted on anesthetized animals have shown that inhibition of NO synthesis enhanced sympathetic nerve activity. Sakuma et al2 reported that systemic administration of the NO synthase inhibitor L-NMMA increased RSNA in anesthetized rats, which could be completely abolished by spinal C1 to C 2 transection. Togashi et al3 further demonstrated that intracisternal administration of L-NMMA elicited a marked increase in RSNA. These results suggested that endogenous NO inhibited sympathetic outflow in anesthetized rats by a central mechanism. A recent study in anesthetized cats1 also demonstrated that inhibition of peripheral sympathetic vasoconstriction was an important mechanism for the vasodilation induced by NO in vivo. In contrast, in the present study, we found that intravenous infusion of L-NAME increased MAP and decreased RSNA. Considering that a rise in MAP will activate the baroreceptors and cause a reflex reduction in the sympathetic outflow, which might obscure the sympathoexcitatory effect of L-NAME, we returned the MAP to the pre–L-NAME level by intravenous infusion of hydralazine in order to eliminate the potentially confounding influence of baroreflex activation. We failed to see a sympathoexcitatory effect of L-NAME even when the influence of baroreflex activation was eliminated. L-NAME has been widely used as a specific NO synthesis inhibitor at the dose used in the present study.21 Thus, the failure to observe sympathoexcitation after NO synthase blockade in the present study was not due to the specific analogue we chose. In a related study from this laboratory8 carried out in conscious rabbits, we also failed to observe an increase in RSNA after an intravenous infusion of another NO synthase inhibitor, L-NNA. Our results are consistent with a recent study by Hansen et al9 carried out in healthy humans in which they could not observe an increase in muscle sympathetic nerve activity after blockade of endogenous NO synthesis by L-NMMA.
The apparent contradictory findings between the studies performed in anesthetized rats and cats and those carried out in conscious rabbits and humans might be due to several factors. One factor is a species difference. It has been reported that microinjection of an NO synthase inhibitor into the RVLM increased sympathetic outflow in anesthetized cats7 but decreased sympathetic outflow in anesthetized rabbits.22 Another factor that might contribute to the differences is the experimental setting. Sigmon and Beierwaltes23 reported that anesthesia increased plasma renin activity and circulating Ang II, which resulted in an increased interaction between the vasodilating effects of NO and the vasoconstricting effects of Ang II in regulating regional hemodynamics. In the present study, L-NAME increased RSNA in conscious rabbits only during a background infusion of Ang II. This suggests that a high level of Ang II is critical in order to observe the sympathoexcitatory effect of NO synthase inhibition. This finding might provide an explanation for the discrepancies between the results of experiments carried out in anesthetized versus conscious subjects.
Increasing evidence has suggested that there is a close interaction between NO and Ang II on a variety of tissues. Ang II increased NO release in many cell types, including cultured murine neuroblastoma cells,24 vascular smooth muscle cells, and cardiac cells.25 26 The basal and stimulated release of NO counteracted the responses induced by Ang II,27 28 whereas blockade of NO synthesis significantly enhanced the vasoconstrictor effects of Ang II.29 30 On the other hand, Qiu et al31 reported that Ang II mediated the hypertensive effect induced by blockade of NO synthesis. It has also been shown that Ang II and NO exerted opposite effects in the central nervous system.15 32 The present data further demonstrate that there is a major impact of NO and Ang II on the regulation of sympathetic tone.
Several studies have shown that Ang II itself has sympathoexcitatory effects.33 However, the augmented RSNA after NO synthesis blockade with a background infusion of Ang II observed in the present study was not due to the direct effects of Ang II on sympathetic nerve activity, since Ang II alone did not alter RSNA at the dose used in the present study. It is also unlikely that this effect was secondary to the pressor action induced by Ang II, because we failed to see an increase in RSNA when substituting an equipressor dose of PE for Ang II.
The functional Ang II receptors involved in this response appear to be predominantly of the AT1 subtype. Recent evidence has suggested an important role for the central AT1 receptor in modulating baroreflex control of RSNA in rats with congestive heart failure.34 In the present study, after administration of the selective AT1 receptor antagonist losartan, the enhancement of RSNA induced by NO synthase blockade with a background infusion of Ang II was completely abolished.
It should be noted that alkyl esters of l-arginine, such as L-NAME, are antagonists of muscarinic cholinergic receptors.35 To eliminate the possibility that the sympathoexcitation induced by L-NAME with a background infusion of Ang II was due to its antimuscarinic effect, another arginine analogue, L-NNA, was substituted for L-NAME in two additional rabbits. The results showed that blockade of NO synthesis with L-NNA induced responses similar to those produced by L-NAME. The specificity of the effects of L-NAME was further tested by comparing the responses to D-NAME. Under the same conditions, D-NAME had no effects on MAP and RSNA.
Although the present study does not provide direct evidence for the site of action of the NO–Ang II interaction, we propose that this is mediated via a central mechanism. Ang II has been reported to act at several central sites that are known to alter sympathetic outflow. One important site is the area postrema, which is accessible to circulating Ang II.33 This circumventricular organ sends projections to structures such as the NTS, the DMV, the RVLM, and the lateral parabrachial nuclei, all of which play important roles in the regulation of sympathetic nerve activity.36 Moreover, NO synthase has been demonstrated to be present in the NTS, the RVLM, and the DMV.5 22 37 38 L-NAME can inhibit NO synthase in the brain, since it has been shown that intravenous administration of L-NAME elicited a prolonged inhibition of NO synthase.21 Harada et al5 reported that inhibition of NO formation in the NTS increased renal sympathetic nerve activity in rabbits. However, Hirooka et al22 showed that microinjection of L-NAME into the RVLM elicited a decrease in RSNA in rabbits. It is possible that NO exerts opposite influences on sympathetic nerve activity at different sites that normally are inhibitory to each other. This may explain the failure to see sympathoexcitation after systemic administration of L-NAME. It is possible that during a background infusion of Ang II, the balance of the sympathoinhibitory effect and the sympathoexcitatory effect induced by endogenous NO at different central sites may be shifted toward the sympathoexcitatory state. NO might function as a negative-feedback modulator of sympathetic nerve activity. Therefore, blockade of NO should have a prominent sympathoexcitatory effect when Ang II is elevated.
Limitations of the Study
Although these data strongly suggest that Ang II mediates sympathoexcitation in the face of NO synthesis blockade, there are two concerns that should be addressed. First, Ang II in the present study was administered to the peripheral circulation, and we have no evidence that an increase in plasma Ang II is reflected by an increase in central Ang II. Although we did not measure plasma or brain Ang II in the present study, there is good evidence that peripheral Ang II can gain entry into the brain through those areas that lack a blood-brain barrier.33 39
Although we did not measure plasma levels of Ang II during the infusion, the increase in arterial pressure was modest and less than that reported by Kumagai and Reid40 for the same infusion rate in conscious rabbits. On the basis of data reported by Brooks et al41 and by Reid and Chou,42 we estimate the plasma concentration of Ang II after our infusion to be <100 pg/mL, a significant increase from normal (≈10 to 20 pg/mL).42 Therefore, the plasma levels in the present study are most likely within the range seen in states in which the renin–Ang II system is activated.
One of the premises behind the present study is that the increase in RSNA after NO synthesis blockade in anesthetized preparations may be due to increased plasma levels of Ang II. Although we do not have data on Ang II concentration in conscious versus anesthetized rabbits, we would estimate from data on anesthetized rats43 that plasma Ang II in anesthetized rabbits is >100 pg/mL, significantly higher than in conscious rabbits.39
Hirai et al44 have shown that NO synthase inhibition in anesthetized rats can produce nonuniform changes in RSNA and lumbar sympathetic nerve activity. L-NAME augmented RSNA but attenuated lumbar sympathetic nerve activity. Only RSNA was recorded in the present study. Whether the effect of L-NAME on RSNA observed in the present study can be extrapolated for sympathetic outflow to other beds needs to be further examined. However, because Hansen et al9 failed to see augmentation of muscle sympathetic nerve activity after the administration of L-NMMA to conscious humans, it is unlikely that we would observe different responses in skeletal muscle outflow in our preparation.
In summary, we found that in conscious rabbits, blockade of NO synthesis alone had no independent effect on sympathetic nerve activity. However, during a background infusion of Ang II, there was a significant increase in sympathetic nerve activity after blockade of NO synthesis. The present study demonstrates an important modulating effect of NO on the sympathoexcitatory effect of Ang II. These results provide a possible mechanism for the increase in sympathetic outflow in disease states in which the renin–Ang II system is activated and in which NO synthesis is decreased, such as in chronic severe congestive heart failure.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1||=||Ang II type 1|
|D-NAME||=||Nω-nitro-d-arginine methyl ester|
|DMV||=||dorsal motor nucleus of the vagus|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|MAP||=||mean arterial blood pressure|
|NTS||=||nucleus tractus solitarius|
|RSNA||=||renal sympathetic nerve activity|
|RVLM||=||rostral ventrolateral medulla|
This study was supported by National Heart, Lung, and Blood Institute grant HL-38690. Dr Murakami was a Fellow of the Nebraska Heart Association. The authors wish to thank Johnnie F. Hackley and Pamela Curry for their expert technical assistance. We also thank Dr Kashinath D. Patil for his assistance with the statistical analysis used in this study.
- Received August 22, 1997.
- Accepted December 3, 1997.
- © 1998 American Heart Association, Inc.
Zanzinger J, Czachurski J, Seller H. Inhibition of sympathetic vasoconstriction is a major principle of vasodilation by nitric oxide in vivo. Circ Res. 1995;75:1073–1077.
Sakuma I, Togashi H, Yoshioka M, Saito H, Yanagida M, Tamura M, Kobayashi T, Yasuda H, Gross SS, Levi R. NG-Methyl-l-arginine, an inhibitor of l-arginine–derived nitric oxide synthesis, stimulates renal sympathetic nerve activity in vivo: a role for nitric oxide in the central regulation of sympathetic tone. Circ Res. 1992;70:607–611.
Togashi H, Sakuma I, Yoshioka M, Kobayashi T, Yasuda H, Kitabatake A, Saito H, Gross SS, Levi R. A central nervous system action of nitric oxide in blood pressure regulation. J Pharmacol Exp Ther. 1992;262:343–347.
Harada S, Tokunaga S, Momohara M, Masaki H, Tagawa T, Imaizumi T, Takeshita A. Inhibition of nitric oxide formation in the nucleus tractus solitarius increases renal sympathetic nerve activity in rabbits. Circ Res. 1993;72:511–516.
Horn T, Smith PM, McLaughlin BE, Bauce L, Marks GS, Pittman QJ, Ferguson AV. Nitric oxide actions in paraventricular nucleus: cardiovascular and neurochemical implications. Am J Physiol. 1994;266:R306–R313.
Zanzinger J, Czachurski J, Seller H. Inhibition of basal and reflex-mediated sympathetic activity in the RVLM by nitric oxide. Am J Physiol. 1995;268:R958–R962.
Liu J-L, Murakami H, Zucker IH. Effects of NO on baroreflex control of heart rate and renal nerve activity in conscious rabbits. Am J Physiol. 1996;270:R1361–R1370.
Hansen J, Jacobsen TN, Victor RG. Is nitric oxide involved in the tonic inhibition of central sympathetic outflow in humans? Hypertension. 1994;24:439–444.
Alberola AM, Salazar FJ, Nakamura T, Granger JP. Interaction between angiotensin II and nitric oxide in control of renal hemodynamics in conscious dogs. Am J Physiol. 1994;267:R1472–R1478.
Sigmon DH, Beierwaltes WH. Renal nitric oxide and angiotensin II interaction in renovascular hypertension. Hypertension. 1993;22:237–242.
Zanchi A, Schaad NC, Osterheld M-C, Grouzmann E, Nussberger J, Brunner HR, Waeber B. Effects of chronic NO synthase inhibition in rats on renin-angiotensin system and sympathetic nervous system. Am J Physiol. 1995;268:H2267–H2273.
Kumagai H, Averill DB, Khosla MC, Ferrario CM. Role of nitric oxide and angiotensin II in the regulation of sympathetic nerve activity in spontaneously hypertensive rats. Hypertension. 1993;21:476–484.
Haberl RL, Decker PJ, Einhaupl KM. Angiotensin degradation products mediate endothelium-dependent dilation of rabbit brain arterioles. Circ Res. 1991;68:1621–1627.
Faber JE. Effects of althesin and urethan-chloralose on neurohumoral cardiovascular regulation. Am J Physiol. 1989;256:R757–R765.
Sigmon DH, Beierwaltes WH. Angiotensin II: Nitric oxide interaction and the distribution of blood flow. Am J Physiol. 1993;265:R1276–R1283.
Chaki S, Inagami T. New signaling mechanism of angiotensin II in neuroblastoma Neuro-2A cells: activation of soluble guanylyl cyclase via nitric oxide synthesis. Mol Pharmacol. 1993;43:603–608.
Seyedi N, Xu X, Nasjletti A, Hintze TH. Coronary kinin generation mediates nitric oxide release after angiotensin receptor stimulation. Hypertension. 1995;26:164–170.
Ikeda U, Maeda Y, Kawahara Y, Yokoyama M, Shimada K. Angiotensin II augments cytokine-stimulated nitric oxide synthesis in rat cardiac myocytes. Circulation. 1995;92:2683–2689.
Schnackenberg CG, Wilkins MR, Granger JP. Role of nitric oxide in modulation of the vasoconstrictor actions of angiotensin II in preglomerular and postglomerular vessels in dogs. Hypertension. 1995;26:1024–1029.
Ohishi K, Carmines PK, Inscho EW, Navar LG. EDRF-angiotensin II interactions in rat juxtamedullary afferent and efferent arterioles. Am J Physiol. 1992;263:F900–F906.
Qiu C, Engels K, Baylis C. Angiotensin II and α1-adrenergic tone in chronic nitric oxide blockade-induced hypertension. Am J Physiol. 1994;266:R1470–R1476.
Schmid HA, Schafer F, Simon E. Opposite effects of angiotensin II and nitric oxide on neurons in the duck subfornical organ. Neurosci Lett. 1995;187:149–152.
Reid IA. Interactions between ANG II, sympathetic nervous system, and baroreceptor reflexes in regulation of blood pressure. Am J Physiol. 1992;262:E763–E778.
Dibona GF, Jones SY, Brooks V. ANG II receptor blockade and arterial baroreflex regulation of renal nerve activity in cardiac failure. Am J Physiol. 1995;269:R1189–R1196.
Buxton ILO, Cheek DJ, Eckman D, Westfall DP, Sanders KM, Keef KD. NG-Nitro l-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists. Circ Res. 1993;72:387–395.
Matsukawa S, Reid IA. Role of the area postrema in the modulation of the baroreflex control of heart rate by angiotensin II. Circ Res. 1990;67:1462–1473.
Travagli RA, Gillis RA. Nitric oxide-mediated excitatory effect on neurons of dorsal motor nucleus of vagus. Am J Physiol. 1994;266:G154–G160.
Kumagai K, Reid IA. Angiotensin II exerts differential actions on renal nerve activity and heart rate. Hypertension. 1994;24:451–456.
Brooks VL, Ell KR, Wright RM. Pressure-independent baroreflex resetting produced by chronic infusion of angiotensin II in rabbits. Am J Physiol. 1993;265:H1275–H1282.
Huang H, Baussant T, Reade R, Michel JB, Corvol P. Measurement of angiotensin II concentration in rat plasma: pathophysiological applications. Clin Exp Hypertens. 1989;A11:1535–1548.
Hirai T, Musch TI, Morgan DA, Kregel KC, Claassen DE, Pickar JG, Lewis SJ, Kenney MJ. Differential sympathetic nerve responses to nitric oxide synthase inhibition in anesthetized rats. Am J Physiol. 1995;269:R807–R813.