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Am J Physiol Regul Integr Comp Physiol 290: R741-R748, 2006. First published October 27, 2005; doi:10.1152/ajpregu.00660.2005
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Role of nitrosyl factors in the hindlimb vasodilation elicited by baroreceptor afferent nerve stimulation

Olga S. Possas,1 Alan Kim Johnson,1,2,3 and Stephen J. Lewis4

Departments of 1Pharmacology and 2Psychology and 3The Cardiovascular Center, University of Iowa, Iowa City, Iowa; and 4Department of Physiology and Pharmacology, University of Georgia, Athens, Georgia

Submitted 9 September 2005 ; accepted in final form 26 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study determined whether electrical stimulation (ES) of the baroreceptor afferent fibers in the aortic depressor nerve (ADN) produces hindlimb vasodilation in pentobarbital-anesthetized rats via the release of nitric oxide (NO)-containing (nitrosyl) factors from NO synthase-positive lumbar sympathetic nerve terminals. ES of the ADN (1–10 Hz for 15 s) produced frequency-dependent reductions in mean arterial blood pressure (MAP) and mesenteric and hindlimb vascular resistance (MR and HLR, respectively). The falls in resistance were substantially smaller in hindlimb beds in which the ipsilateral lumbar sympathetic chain had been transected 7–10 days previously. The maximal falls in MR and hindquarter vascular resistance (HQR) produced by 1- to 10-Hz ES of the ADN were unaffected by the specific inhibitor of neuronal NO synthase 7-nitroindazole (7-NI, 45 mg/kg iv). However, the total falls in HQR (mmHg·kHz–1·s) produced by these stimuli were significantly diminished by 7-NI, whereas the total falls in MR were not affected. Four successive episodes of 10-Hz ES produced equivalent reductions in MAP, MR, and HQR. The peak changes in these parameters were not affected by 7-NI. However, the total falls in HQR progressively diminished with each successive stimulus, whereas the total falls in MR remained unchanged. These results provide evidence that the hindlimb vasodilation produced by ES of baroreceptor afferents within the ADN may involve the activation of postganglionic lumbar sympathetic vasodilator fibers, which release newly synthesized and preformed nitrosyl factors.

aortic depressor nerve; nitric oxide; rat

THERE IS SUBSTANTIAL EVIDENCE that an active neurogenic vasodilator system innervates the skeletal musculature of the limbs (1–9, 18, 20, 22, 33). The pharmacological (22), physiological (3, 12, 15), or direct electrical activation (5, 8, 13, 14) of nerves in sympathetic trunks innervating the forearm or hindlimb elicits pronounced vasodilator responses. The noncholinergic, nonadrenergic vasodilator system may partially involve the release of histamine in some species (1, 9). It is also possible that these nerves elicit vasodilation indirectly via the release of endothelium-derived relaxing factors (12–15). It is also possible that the nerves innervating the hindlimb vasculature directly release vasodilator factors, which have not been conclusively identified.

There is evidence that postganglionic lumbar sympathetic cell bodies of the rat contain a constitutive form of nitric oxide (NO) synthase (NOS), that NOS undergoes axonal transport toward the peripheral terminals in hindlimb vessels, and that vascular smooth muscle of iliac and femoral arteries contains neuronal varicosities that stain densely for NOS (13). In addition, the direct (13, 14) and centrally mediated (12, 15) activation of the lumbar sympathetic chain produces a pronounced hindlimb vasodilation in pentobarbital-anesthetized rats that may be due to the release of newly synthesized and preformed NO-containing factors (NOFs) from NOS-positive nerve terminals.

The electrical stimulation (ES) of the superior laryngeal nerve (SLN) produces frequency-dependent falls in hindquarter vascular resistance (HQR) in pentobarbital-anesthetized rats (16, 31). The hindquarter vasodilation is largely dependent on the integrity of the lumbar sympathetic chain (16, 31) and may involve the release of NOFs from NOS-positive sympathetic nerve terminals (31). The SLN contains mechanosensitive, chemosensitive, and baroreceptor afferent fibers (4, 16, 19, 31). Therefore, it was not possible to determine which afferent fiber type within the SLN may have been responsible for the activation of the lumbar sympathetic vasodilator system (31). In contrast, the aortic depressor nerve (ADN) carries baroafferent fibers only (35). In anesthetized rats, direct ES of the ADN elicits pronounced falls in HQR (25) that involve the withdrawal of sympathetic neurogenic vasoconstriction. However, there is considerable evidence that the physiological activation of baroreceptor afferents also produces hindlimb vasodilation by the activation of a sympathetic neurogenic vasodilator system (1, 2, 9, 18, 22).

The aim of this study was to examine whether the hindlimb vasodilation elicited by ES of the ADN in pentobarbital-anesthetized rats may involve the activation of NOS-positive postganglionic sympathetic nerves, which release newly synthesized and preformed stores of NOFs.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rats. All studies were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 80-23, Revised 1996]. All protocols were approved by the University of Iowa Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (250–300 g, n = 39; Harlan, Madison, WI) were used in these studies.

Transection of the lumbar sympathetic chain. Rats were anesthetized with acepromazine maleate (12 mg/kg ip) and ketamine (120 mg/kg ip). A midline laparotomy was performed, and the left lumbar sympathetic chain was isolated, cut, and removed caudally to the bifurcation of the left common iliac artery and vein (11, 15, 31). The right sympathetic chain was left intact. In sham-operated rats, the left lumbar sympathetic chain was isolated but not cut. The rats were allowed 7–10 days to recover from the surgeries.

Catheterization and placement of Doppler flow probes. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and catheters (PE-50) were placed into the left common carotid artery and jugular vein for measurement of pulsatile pressure (PP) and mean arterial blood pressure (MAP) and administration of drugs, respectively. Once the venous catheter was in place, pentobarbital sodium (5 mg/kg iv) was given every 30 min. In the rats in which the role of the lumbar sympathetic nerves in the hindlimb vasodilation produced by ES of the ADN was to be examined, miniature pulsed Doppler flow probes were placed on the superior mesenteric and left and right iliac arteries for measurement of mesenteric blood flow (MF) and hindlimb blood flow (HLF) and determination of mesenteric and left and right hindlimb vascular resistances (MR, HLRL, and HLRR, respectively) (31). In other experiments, Doppler flow probes were placed on the superior mesenteric artery and the descending aorta for measurement of MF and hindquarter blood flow (HQF) and determination of MR and HQR (31). Vascular resistance at any point in time was determined by the following formula: vascular resistance = MAP ÷ blood flow (17). The area under the curve (AUC, mmHg·kHz–1·s) for changes in HQR was calculated by enlargement of the traces by photocopying to allow resistance values to be determined for every second for the entire response. The values were added together to produce the total AUC. Details of the Doppler technique, including construction of the probes, reliability of the method for estimation of flow velocity, and quantitative determination of percent changes in vascular resistances have been described elsewhere (17). The pulsed Doppler flow method does not allow completely accurate animal-to-animal comparisons of absolute flow rate (17). The arterial catheter was connected to a Beckman dynograph-coupled pressure transducer to measure PP and MAP. The wire leads from the flow probes were connected to a Beckman dynograph-coupled Doppler flowmeter for measurement of blood flow velocities.

Isolation and ES of the ADN. A midline incision was made on the ventral surface of the neck, and the left omohyoid muscle was cut and reflected to expose the left ADN. A 4- to 6-mm length of the nerve, beginning from its point of emergence from the SLN, was isolated under a dissecting microscope. The ADN was placed on a bipolar platinum electrode with an interelectrode distance of 1 mm. The nerve and electrode were raised above the surrounding tissue and bathed in warm mineral oil. Square-wave 1-, 2.5-, 5-, 7.5-, and 10-Hz ES (8 V, 0.5 ms) was applied to the ADN for 15 s. The ES was delivered by a Grass S-4 stimulator via a stimulus isolation unit. Sufficient time (5–10 min) was allowed between each stimulus to allow the hemodynamic parameters to return to control values.

Protocol 1: role of the lumbar sympathetic chain in responses elicited by ES of the ADN. The effects of 1- to 10-Hz ES of the ADN for 15 s on heart rate, MAP, MR, HLRL, and HLRR were examined in sham-operated rats (n = 7) and in rats in which the left lumbar sympathetic chain was transected (n = 7). At the end of these experiments, the effectiveness of the denervation was determined by examining the vasodilator effects of the {alpha}1-adrenoceptor antagonist prazosin (100 µg/kg iv) (11, 15, 31).

Protocol 2: effects of the neuronal NOS inhibitor 7-nitroindazole on responses elicited by ES of the ADN. The effects of one episode of 1-, 2.5-, 5-, and 7.5-Hz ES of the ADN (8 V, 0.5 ms for 15 s) and then four to six episodes of 10-Hz ES (8 V for 15 s) on hemodynamic parameters were examined before and 30 min after intravenous injection of vehicle (n = 6) or the specific inhibitor of neuronal NOS 7-nitroindazole (7-NI, 45 mg/kg iv, n = 7) (28, 29, 31). In each case, the hemodynamic responses were allowed to fully recover before the next ES was applied.

Protocol 3: effects of the neuronal/endothelial NOS inhibitor NG-nitro-L-arginine methyl ester on responses elicited by ES of the ADN. Rats were prepared as described for the 7-NI studies. The effects of one episode of 1- to 10-Hz ES of the ADN (8 V, 0.5 ms for 15 s) on MAP, MR, and HQR were examined before and 30 min after intravenous administration of saline (0.9% NaCl, n = 6) or NG-nitro-L-arginine methyl ester (L-NAME, 50 µmol/kg, n = 6), which blocks neuronal and endothelial forms of NOS (12–15, 27, 32).

Drugs. All drugs, except 7-NI, were obtained from Sigma (St. Louis, MO). 7-NI (Color Your Enzyme, Ontario, Canada) was dissolved in Na2CO3 (8% wt/vol at 80°C) (31). The solution was cooled to 37°C and adjusted to pH 7.4 by the addition of 1.0 M HCl. Vehicle solutions consisted of Na2CO3 (8% wt/vol) at pH 7.4 (31).

Statistics. Values are means ± SE. All data were analyzed by repeated-measures ANOVA (37) followed by Student's modified t-test with Bonferroni's correction for multiple comparisons between means using the modified error mean square term from the ANOVA (36). P < 0.05 was taken to denote a statistical difference.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Role of the lumbar sympathetic chain in responses produced by ES of the ADN. The resting parameters in sham-operated rats or in rats in which the left lumbar sympathetic trunk had been transected 7–10 days previously are summarized in Table 1. MAP and MR values were similar in both groups. HLRL and HLRR were similar in the sham-operated group. In contrast, resting resistances were higher in the sympathetically denervated than in the nondenervated hindlimbs (HLRR). Resting resistances in the nondenervated hindlimbs were similar in both groups (P > 0.05). The effects of 1- to 10-Hz ES of the ADN on MAP, MR, and HLR in rats in which the left lumbar sympathetic chain had been transected 7–10 days previously are summarized in Fig. 1. ES of the ADN elicited frequency-dependent reductions in MAP, MR, and resistances in the sympathetically intact and sympathetically denervated hindlimb beds. The vasodilator responses in the denervated bed were markedly smaller than those in the innervated bed. Prazosin markedly reduced resistance in the sympathetically intact (–39 ± 4%, P < 0.05) but not in the denervated hindlimb bed (–2 ± 4%, P > 0.05), thus confirming the effectiveness of the surgical sympathectomy. ES of the ADN produced similar falls in resistance in the left and right hindlimbs of the sham-operated rats (n = 7, P > 0.05 for all comparisons; data not shown).


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  		Table 1. Resting hemodynamic parameters is sham-operated and sympathetically denervated rats

 

Figure 1
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  		Fig. 1. Effects of 1- to 10-Hz electrical stimulation (ES, 0.5 ms for 15 s) of aortic depressor nerve (ADN) on mean arterial blood pressure (MAP), mesenteric vascular resistance (MR), and hindlimb vascular resistance (HLR) in sympathetically intact and sympathetically denervated hindlimbs of pentobarbital-anesthetized rats (n = 7). Value are means ± SE of maximal percent changes. *P < 0.05 vs. intact.

 
Effects of 7-NI on responses to a single episode of ES of the ADN. Resting parameters recorded over the period during which the frequency-response curves were established before and after administration of 7-NI are summarized in Table 2. For each rat in the group, the value before each stimulus was recorded and the mean value was calculated. This mean value from each rat was taken to calculate the mean ± SE values for the group. 7-NI did not alter MAP or MR but did slightly increase resting HQR. Administration of vehicle did not affect resting parameters (n = 6, P > 0.05 for all comparisons). A typical example of the responses elicited by 7.5-Hz ES of the ADN (8 V for 15 s) before and after injection of 7-NI is shown in Fig. 2. First, ES of the ADN elicited a pronounced fall in MAP and an increase in HQF. The changes in MAP and blood flow were sustained during ES of the ADN (a minor reduction in HQF was seen at the termination of the ES). The reductions in MAP and HQF returned to baseline levels after termination of the ES. The maximal fall in MAP was –26%. At this point, HQF was minimally increased. After injection of 7-NI, the ES of the ADN produced a maximal reduction in MAP (28%) similar to that observed before the injection of 7-NI. Again, HQF was minimally changed at this time. The maximal reductions in HQR (i.e., vasodilation) before and after administration of 7-NI were 39% and 28%, respectively. Before administration of 7-NI, the maximal increase in HQF occurred just as MAP began to return to baseline. This increase in HQF was substantially smaller after administration of 7-NI. The reductions in HQR when MAP began to return to baseline (point of maximal increase in HQF) before and after administration of 7-NI were 46% and 21%, respectively. From the entire response, it was evident that 7-NI markedly reduced the total vasodilator response (AUC for flow) in the hindquarter beds. Second, administration of 7-NI did not affect the vasodilator responses in the mesenteric bed. The maximal reductions in MR before and after administration of 7-NI were 15% and 16%, respectively.


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  		Table 2. Resting hemodynamic variables recorded over electrical stimulation period before and after administration of 7-NI

 

Figure 2
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  		Fig. 2. Effects of 7.5-Hz ES of ADN (8 V, 0.5 ms for 15 s) on pulsatile pressure (PP), MAP, and mesenteric and hindquarter blood flows (MF and HQF, respectively) in a pentobarbital-anesthetized rat before and after injection of 7-nitroindazole (7-NI, 45 mg/kg iv).

 
The maximal responses elicited by ES of the ADN before and after injection of 7-NI are summarized in Fig. 3. ES of the ADN elicited frequency-dependent falls in MAP, MR, and HQR. The maximal responses were not affected by 7-NI. The responses elicited by ES of the ADN were similar before and after injection of vehicle (P > 0.05 for all comparisons). For example, the falls in HQR before injection of vehicle were –3 ± 2%, –12 ± 3%, –29 ± 4%, –35 ± 6%, and –50 ± 9%, respectively. After injection of vehicle, the changes in HQR were –5 ± 3%, –16 ± 3%, –27 ± 6%, –35 ± 6%, and –47 ± 7%, respectively (P > 0.05 for all comparisons between pre- and postvehicle responses at each frequency). As mentioned above, it was evident that the total hindlimb vasodilation was reduced by 7-NI. The total falls in HQR (AUC for the change in HQR over time) elicited by 2.5- to 10-Hz ES of the ADN are summarized in Fig. 4 (top). The total falls in HQR increased with stimulus intensity. The total falls in HQR elicited by ES of the ADN were reduced after injection of 7-NI. In contrast, the total falls in MR elicited by the 2.5- to 10-Hz ES of the ADN were not affected by 7-NI (P > 0.05 for all comparisons; data not shown). In addition, the total falls in HQR and MR produced by 2.5- to 10-Hz ES of the ADN were similar before and after injection of vehicle (P > 0.05 for all comparisons; data not shown).


Figure 3
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  		Fig. 3. Effects of 1- to 10-Hz ES of ADN (8 V, 0.5 ms for 15 s) on MAP, MR, and hindquarter vascular resistance (HQR) in pentobarbital-anesthetized rats (n = 7) before and after injection of 7-NI (45 mg/kg iv). Values are means ± SE.

 

Figure 4
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  		Fig. 4. Top: total hindlimb vasodilation as measured by area under the curve (AUC) for change in HQR over time produced by 2.5- to 10-Hz ES of ADN in pentobarbital-anesthetized rats (n = 7) before and after administration of 7-NI (45 mg/kg iv). Values are means ± SE. *P < 0.05 vs. pre-7-NI at each stimulus level. Bottom: total hindlimb vasodilation produced by 4 successive episodes of 10-Hz ES of ADN before and after administration of 7-NI (45 mg/kg iv). Values are means ± SE. A smaller AUC represents a reduced overall vasodilation compared with that produced by the 1st ES. *P < 0.05 vs. pre-7-NI for each 10-Hz stimulus. {dagger}P < 0.05 vs. 1st ES.

 
Effects of 7-NI on responses elicited by successive episodes of 10-Hz ES of the ADN. Typical examples of the responses elicited by four successive episodes of 10-Hz ES of the ADN (8 V, 0.5 ms for 15 s) before injection of 7-NI are shown in Fig. 5. The first ES elicited a pronounced fall in MAP, a minor fall in MF, and an increase in HQF (followed by a reduction in HQF at the termination of the ES). The reductions in MAP and associated changes in MF and HQF elicited by each ES were very similar to one another. Typical examples of the responses elicited by four successive episodes of 10-Hz ES of the ADN (8 V, 0.5 ms for 15 s) after injection of 7-NI are shown in Fig. 6. The first ES elicited a pronounced fall in MAP, a fall in MF, and an increase in HQF. The increase in HQF was maximal at the point at which MAP began to return to baseline. Each subsequent ES produced similar changes in MAP and MF. Because flow remained constant at the point of maximal reduction in MAP, each ES elicited similar maximal reductions in HQR. However, the maximal increase in HQF gradually diminished with each ES, so that the reductions in HQR at this and subsequent points progressively diminished with each successive ES. Each ES elicited similar changes in MF and, therefore, MR.


Figure 5
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  		Fig. 5. Effects of 4 successive episodes of 10-Hz ES of ADN (8 V, 0.5 ms for 15 s) on PP, and MAP, MF, and HQF in a pentobarbital-anesthetized rat before administration of 7-NI (45 mg/kg iv).

 

Figure 6
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  		Fig. 6. Effects of 4 successive episodes of 10-Hz ES of ADN (8 V, 0.5 ms for 15 s) on PP, MAP, MF, and HQF in a pentobarbital-anesthetized rat after administration of 7-NI (45 mg/kg iv).

 
The maximal responses elicited by successive episodes of 10-Hz ES of the ADN before and after injection of 7-NI are summarized in Fig. 7. Before injection of 7-NI, each episode of ES elicited similar falls in MAP, MR, and HQR. 7-NI did not modify the maximal responses elicited by these stimuli. Four successive episodes of 10-Hz ES of the ADN (8 V for 15 s) elicited similar responses before and after injection of vehicle (P > 0.05 for all comparisons). For example, the falls in HQR produced by the four episodes of ES before injection of vehicle were –44 ± 6%, –41 ± 4%, –41 ± 6%, and –47 ± 8%, respectively (P > 0.05 for all comparisons), whereas after injection they were –47 ± 5%, –43 ± 6%, –41 ± 9%, and –44 ± 6%, respectively (P > 0.05 for all comparisons). The total falls in HQR (AUC for the change in HQR over time) elicited by four successive episodes of 10-Hz ES of the ADN before and after administration of 7-NI are summarized in Fig. 4, bottom. Before injection of 7-NI, each of the four episodes of 10-Hz ES of the ADN produced a similar total fall in HQR. After administration of 7-NI, the total fall in HQR produced by the first episode of ES of the ADN was less than that observed before injection of 7-NI. In addition, the total falls in HQR progressively and substantially diminished with each successive episode of ES of the ADN. In contrast, the total falls in MR produced by the four successive episodes of 10-Hz ES of the ADN were not affected by 7-NI (P > 0.05 for all comparisons; data not shown).


Figure 7
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  		Fig. 7. Effects of 4 successive episodes of 10-Hz ES of ADN (8 V, 0.5 ms for 15 s) on MAP, MR, and HQR in pentobarbital-anesthetized rats (n = 7) before and after administration of 7-NI (45 mg/kg iv). Values are means ± SE. There were no differences in these responses before or after administration of 7-NI (P < 0.05).

 
Effects of L-NAME on responses produced by a single episode of ES of the ADN. The effects of saline or L-NAME (50 µmol/kg iv) on resting parameters are summarized in Table 3. Saline did not affect these parameters, whereas L-NAME elicited substantial increases in MAP, MR, and HQR, which were sustained for the duration of the experiments. The maximal responses elicited by 1- to 10-Hz ES of the ADN before and after injection of L-NAME are summarized in Fig. 8. The maximal frequency-dependent falls in MAP, HQR, and MR produced by ES of the ADN were substantially diminished after administration of L-NAME. In contrast, the maximal hemodynamic responses produced by 1- to 10-Hz ES of the ADN were similar before and after administration of saline (P > 0.05 for all comparisons; data not shown).


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  		Table 3. Resting hemodynamic variables before and 15–60 min after injection of saline or L-NAME

 

Figure 8
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  		Fig. 8. Effects of 1- to 10-Hz ES of ADN (8 V, 0.5 ms for 15 s) on MAP, MR, and HQR in pentobarbital-anesthetized rats (n = 6) before and after administration of NG-nitro-L-arginine methyl ester (L-NAME, 50 µmol/kg iv). Values are means ± SE. *P < 0.05 vs. pre-L-NAME.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study support our previous evidence that nonadrenergic, noncholinergic vasodilator nerves within the lumbar sympathetic chain innervating the hindlimb vasculature of rat release newly synthesized and preformed stores of nitrosyl factors (12–15). The principal novel aspect of the present study is that activation of baroafferent fibers in the ADN recruits this lumbar vasodilator system to dilate resistance arteries in the hindlimb of the anesthetized rat. The presence of this system may help explain why activation of baroafferents in the ADN elicited a much more pronounced vasodilation in the hindlimb bed than the mesenteric bed in the present study and in the study of Machado et al. (25).

In pentobarbital-anesthetized rats, ES of the ADN elicited frequency-dependent falls in MAP that were accompanied by pronounced vasodilator responses in the hindquarter bed and less pronounced vasodilator responses in the mesenteric bed. The hindlimb responses elicited by ES of the ADN were markedly reduced, but not abolished, in rats in which the lumbar sympathetic chain had been transected 7–10 days before experimentation. These findings suggest that the vasodilation in the hindlimb depends, in part, on the integrity of the lumbar sympathetic chain. We have obtained preliminary evidence that the hindlimb vasculature is innervated by vasodilator fibers emanating from the inferior mesenteric and hypogastric ganglia (23). Whether the ES of the ADN leads to the centrally mediated activation of these vasodilator fibers is yet to be determined. The responses elicited by ES of the ADN are somewhat comparable to those found by Machado et al. (25), who reported that ES of the ADN elicited a pronounced vasodilation in the hindquarter bed, but a vasoconstriction in the mesenteric bed, in urethane-anesthetized rats. These contrasting findings may be due to the differences in the anesthetic (urethane vs. pentobarbital) or the method of ES delivery (0.5–30 V at 50 Hz vs. 1–10 Hz at 8 V). The observation that resting resistances were higher in sympathetically denervated hindlimb beds than in intact hindlimbs confirms previous findings in conscious (11, 15) and anesthetized (31) rats. The mechanisms underlying this increase in vascular resistance may involve the loss of the sympathetic vasodilator system, which innervates the hindlimb, as well as a downregulation of endothelial NOS activity (11, 15, 31). The lack of effect of the {alpha}1-adrenoceptor antagonist prazosin on resting vascular resistance in the denervated hindlimb is consistent with our previous findings in conscious (15) and pentobarbital-anesthetized rats (31). These findings suggest that circulating levels of catecholamines in pentobarbital-anesthetized rats may not be high enough to affect vascular tone in the denervated hindlimb bed and/or that the sensitivity of adrenergic receptors to circulating catecholamines is somehow downregulated in the denervated bed.

The maximal hindlimb vasodilation elicited by 1- to 10-Hz ES of the ADN was not affected by 7-NI. However, 7-NI markedly reduced the maximal hindlimb vasodilation elicited by ES of the SLN (31). These findings suggest that the vasodilation elicited by ES of the ADN may be initiated by the withdrawal of sympathetic neurogenic vasoconstrictor drive, rather than the release of newly synthesized NOFs. Moreover, these findings suggest that the hindlimb vasodilation initiated by ES of the SLN may be due to activation of nonbaroreceptor afferents in this nerve (4, 16, 19, 31). However, the total hindlimb vasodilation in response to ES of the ADN was substantially reduced by 7-NI. This suggests that the release of NOFs, possibly from the NOS-positive lumbar sympathetic nerve terminals, contributes to the hindlimb vasodilation. In contrast, the maximal and total vasodilator responses in the mesenteric bed in response to ES of the ADN were not affected by 7-NI. This suggests that the vasodilation in the mesenteric bed in response to ES of the ADN was due to withdrawal of sympathetic drive (31). The lack of effect of 7-NI on the mesenteric vasodilation produced by ES of the ADN also suggests that 7-NI did not interfere with the central processing of baroreceptor afferent information. The dose of 7-NI used in the present study (45 mg/kg iv) is comparable to that used by Moore et al. (28, 29), who found that an intraperitoneal injection of 50 mg/kg of 7-NI produced a virtually complete blockade of antinociceptive activity in mice. Moore et al. (29) also reported that pretreatment of mice with L-arginine (50 mg/kg ip) partially attenuated the effects of 7-NI (25 mg/kg ip). We have not determined whether L-arginine attenuates the inhibitory effects of 7-NI on the hindlimb vasodilation produced by ES of the ADN. However, we have reported that L-arginine (500 µmol/kg iv plus 50 µmol·kg–1·min–1 iv) partially attenuated the inhibitory effects of 7-NI on the hindlimb vasodilation produced by ES of the lumbar sympathetic chain in pentobarbital-anesthetized rats (31).

In contrast to the above-mentioned findings, it was evident that L-NAME inhibited the hypotensive and mesenteric and hindquarter vasodilator responses produced by ES of the ADN. L-NAME also significantly attenuated the hemodynamic responses produced by ES of the SLN (31). These findings suggest that systemic L-NAME enters the brain and interferes with the central processing of afferent input resulting from ES of the SLN and ADN. L-NAME is highly lipophilic (11) and, therefore, may be able to gain access to central sites involved in processing the afferent information arising from the ES of these afferent nerves. Moreover, it is possible that 7-NI is not lipophilic enough to gain access to these brain sites. Ma et al. (24) reported that systemic injections of L-NAME (10 mg/kg iv) directly influenced the activity of neurons within the nucleus tractus solitarius (NTS). The majority of neurons were inhibited by L-NAME, and this inhibition was reversed by systemic administration of L-arginine or the NO donor glyceryl trinitrate. Moreover, Ma et al. found that superfusion of rat brain slices with L-NAME decreased the firing rate of neurons in the NTS and that this inhibition was again reversed by L-arginine or glyceryl trinitrate. Therefore, it is possible that the loss of hindlimb vasodilation produced by ES of the ADN and SLN in 7-NI-treated rats may be due to the actions of the NOS inhibitor in the NTS or other structures in the central nervous system. If this were true, then inhibition of NOS in these structures would have to selectively affect autonomic outflow to the hindlimb (see below).

The lack of effect of 7-NI on resting MAP and MR suggests that this compound did not inhibit endothelial NOS, because inhibition of this enzyme elicits pronounced elevations of MAP via increases in vascular resistance (27). Moreover, the lack of effect of 7-NI on MAP and MR suggests that 7-NI did not affect the activity of central or autonomic neurons involved in the regulation of cardiac output or vascular resistance. However, 7-NI slightly increased resting HQR. This finding suggests that the NOS-containing nerves innervating the resistance vessels of the hindlimb vasculature play a role in the maintenance of vasomotor tone. This latter finding differs somewhat from our previous observation that 7-NI produced an increase in resting HQR that did not reach statistical significance (31). However, we have now conducted experiments with 7-NI in a total of 45 rats and found that the 7-NI-induced increase in HQR is similar in magnitude to that reported in the present study.

The finding that the maximal hindlimb vasodilator responses elicited by four episodes of 10-Hz ES of the ADN were similar to one another in 7-NI-treated rats differs from the results obtained with ES of the SLN (31). In one group of 7-NI-treated rats, the first episode of 10-Hz ES of the SLN elicited a fall in HQR that was similar to the fall before injection of 7-NI. However, each subsequent 10-Hz ES of the SLN elicited progressively and substantially smaller vasodilator responses in the hindlimb bed. In another group of 7-NI-treated rats, the maximal hindlimb vasodilation did not progressively decrease with each 10-Hz ES of the SLN, whereas the total hindlimb vasodilation did progressively decrease. The findings in this latter group of rats are similar to those observed in the present study. More specifically, it was evident that the total hindlimb vasodilation produced by 10-Hz ES of the ADN progressively diminished with each successive stimulus. We have no definitive explanation for the somewhat different responses of the two groups of rats in the SLN study (31), nor can we explain why the total, rather than the maximal, hindlimb vasodilation produced by ES of the ADN progressively diminished in the presence of 7-NI. The most obvious explanation is that the hindlimb vasodilation produced by ES of the ADN is initiated by withdrawal of sympathetic vasoconstrictor nerve activity and that this withdrawal of neurogenic vasoconstriction is similar with each 10-Hz stimulus. In contrast, the total vasodilation may progressively decrease because of a gradual depletion of neurogenic stores of nitrosyl factors. The hindquarter vasodilator effects of the endothelium-dependent agonist acetylcholine and the putative endothelium-derived S-nitrosothiol L-S-nitrosocysteine (30) are augmented after administration of 7-NI (31). Therefore, it is unlikely that the 7-NI-induced loss of hindlimb vasodilation produced by ES of the ADN is due to diminished vasodilator potencies of neurogenic- or endothelium-derived nitrosyl factors.

Taken together, these results suggest that activation of baroafferents in the ADN produces vasodilation in the hindlimb beds by two distinct mechanisms. The first mechanism probably involves the withdrawal of sympathetic vasoconstrictor tone, because ES of the ADN reduces postganglionic lumbar sympathetic nerve activity (LSNA) (34, 35). Accordingly, it is unlikely that the vasodilation caused by ES of the ADN is due to the release of catecholamines or ATP from sympathetic nerve terminals, which exert their vasodilator effects via direct actions on vascular smooth muscle and/or release of endothelium-derived NOFs (10, 11, 21, 26). The second mechanism by which ES of the ADN may cause hindlimb vasodilation is activation of lumbar noncholinergic sympathetic vasodilator fibers (1, 2, 9, 18, 22), which use newly synthesized and preformed neurotransmitter stores of NOFs. The likelihood that these fibers are noncholinergic is supported by evidence that hindlimb vasodilation produced by ES of the ADN is not attenuated by the cholinergic muscarinic receptor antagonist methylatropine (25). The maximal baroreflex-mediated reductions in LSNA (i.e., 50–60%) are smaller than the reductions in adrenal and renal sympathetic nerve activity (i.e., 85–95%) (34). Perhaps because baroafferent activation does not inhibit all the preganglionic motor nerves supplying postganglionic nerves in the lumbar trunk, the effects on LSNA are smaller. Specifically, the baroreflex may inhibit fewer vasoconstrictor sympathetic nerve fibers in the lumbar than in the renal or adrenal sympathetic trunks. However, these findings also raise the possibility that activation of the baroreflex increases the activity of a subpopulation of postganglionic lumbar sympathetic vasodilator fibers. Accordingly, the net change in LSNA would be the sum of the inhibition of vasoconstrictor fiber activity and activation of vasodilator fiber activity.

In summary, this study provides evidence that ES of the ADN elicits vasodilation in the hindquarter beds of pentobarbital-anesthetized rats, in part, by activation of a lumbar sympathetic vasodilator system, which utilizes newly synthesized and preformed NOFs. Taken together, these findings support existing evidence that NOFs may serve a neurotransmitter or neuromodulator role in lumbar sympathetic nerves innervating the hindlimb vasculature of the rat (11–15, 31).


   GRANTS
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 ABSTRACT
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 DISCUSSION
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This work was supported in part by National Institutes of Health Grants HL-14388, HL-57472, and DK-66086, National Aeronautics and Space Administration Grant NAG5-6171, and Office of Naval Research Grant N00014-97-1-0145.


   ACKNOWLEDGMENTS
 
Present address of O. S. Possas: Department of Physiology, Federal University of São Paulo (UNIFESP/EPM), Rua Botucatu, 862 V. Clementino, São Paulo, SP 04023-060, Brazil (e-mail: possas@fcr.epm.br).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. K. Johnson, Dept. of Psychology, Univ. of Iowa, 11 Seashore Hall E, Iowa City, IA 52242-1407 (e-mail: alan-johnson{at}uiowa.edu)

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


   REFERENCES
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 DISCUSSION
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