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-adrenergic vasoconstriction in contracting human skeletal muscle
1Department of Health and Exercise Science, Colorado State University, Fort Collins, Colorado 80523-1582; and 2Department of Anesthesiology and General Clinical Research Center, Mayo Clinic and Foundation, Rochester, Minnesota 55905
Submitted 23 June 2004 ; accepted in final form 15 July 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-adrenergic vasoconstrictor responses are blunted in the vascular beds of contracting muscle (functional sympatholysis). We tested the hypothesis that combined inhibition of nitric oxide (NO) and prostaglandins (PGs) restores sympathetic vasoconstriction in contracting human muscle. We measured forearm blood flow via Doppler ultrasound and calculated the reduction in forearm vascular conductance in response to -adrenergic receptor stimulation during rhythmic handgrip exercise (6.4 kg) and during a control nonexercise vasodilator condition (using intra-arterial adenosine) before and after combined local inhibition of NO synthase (NOS; via NG-nitro-L-arginine methyl ester) and cyclooxygenase (via ketorolac) in healthy men. Before combined inhibition of NO and PGs, the forearm vasoconstrictor responses to intra-arterial tyramine (which evoked endogenous noradrenaline release), phenylephrine (a selective 1-agonist), and clonidine (an 2-agonist) were significantly blunted during exercise compared with adenosine treatment. After combined inhibition of NO and PGs, the vasoconstrictor responses to all -adrenergic receptor stimuli were augmented by 
10% in contracting muscle (P < 0.05), whereas the responses to phenylephrine and clonidine were also augmented by 10% during passive vasodilation in resting muscle (P < 0.05). In six additional subjects, PG inhibition alone did not alter the vasoconstrictor responses in resting or contracting muscles. Thus in light of our previous findings, it appears that inhibition of either NO or PGs alone does not affect functional sympatholysis in healthy humans. However, the results from the present study indicate that combined inhibition of NO and PGs augments -adrenergic vasoconstriction in contracting muscle but does not completely restore the vasoconstrictor responses compared with those observed during passive vasodilation in resting muscle.
nitric oxide; prostaglandin; exercise; blood flow; sympathetic nervous system
Although it is clear that sympathetic -adrenergic vasoconstrictor responses can be blunted during muscle contractions, the mechanisms involved in functional sympatholysis in humans are less clear. Substances released from both active muscle and vascular endothelium that have been demonstrated to blunt sympathetic vasoconstriction in a variety of experimental conditions include adenosine (29), prostaglandins (PGs; Refs. 13, 24), and nitric oxide (NO; Ref. 30). In contrast with recent work on experimental animals that indicated a role for NO in functional sympatholysis during exercise (47, 48), our laboratory demonstrated that NO is not obligatory to observe functional sympatholysis in healthy humans (11). This finding has led us to postulate that there are redundant mechanisms capable of blunting sympathetic vasoconstriction during muscle contractions in humans, similar to the metabolic vasodilating substances involved in the regulation of exercise hyperemia (4, 20).
Recently, it was demonstrated that acute inhibition of NO synthase (NOS) results in a substantial increase in shear stress-induced prostacyclin synthesis and release (32). Consonant with this concept, flow-induced vasodilation is relatively preserved in skeletal muscle arterioles of endothelial NOS knockout mice due to enhanced release of vasodilator PGs (43). There is also evidence to indicate that acute inhibition of PG synthesis can result in a compensatory elevation in NO (2) thereby providing additional evidence for a unique interplay between these two vasodilator substances. Therefore, our previous findings indicating the lack of a role for NO in functional sympatholysis in humans might have been "masked" by a compensatory increase in vasodilating PGs during NOS inhibition that subsequently continued to blunt sympathetic vasoconstriction. Given that both NO and PGs have been demonstrated to blunt sympathetic vasoconstrictor responses under certain experimental conditions (13, 24, 30), it seems plausible to speculate that combined inhibition of these substances might augment -adrenergic vasoconstriction in the vascular beds of active muscle.
With this information as a background, the purpose of the present investigation was to test the hypothesis that combined inhibition of NO and PGs restores -adrenergic vasoconstrictor responses in the vascular beds of contracting skeletal muscle in humans. To do so, we measured forearm hemodynamics via Doppler ultrasound during rhythmic handgrip exercise and intra-arterial adenosine infusion (a control vasodilator), and we determined the vasoconstrictor responses to -adrenergic stimulation before and after local NO and PG inhibition. Our findings indicate that acute inhibition of NO and PGs augments postjunctional -adrenergic vasoconstriction, but the combined influence of NO and PGs does not fully account for functional sympatholysis in contracting muscle (i.e., the responses were not completely restored).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A total of 15 young, healthy men [age, 25 ± 2 yr; 79.7 ± 2.9 kg body wt; height, 184 ± 2 cm; body mass index, 23.5 ± 0.8 kg/m2 (means ± SE)] participated in the present study. All were nonsmokers, nonobese, normotensive, and not taking any medications. Studies were performed with the subjects in the supine position after an overnight fast. All studies were approved by the Human Research Committee of the Mayo Clinic and Foundation, and all subjects gave their written informed consent to participate.
Arterial and Venous Catheterization
All subjects received local anesthesia (1% lidocaine) before a 20-gauge, 5-cm catheter was inserted (under aseptic conditions) into the brachial artery of the nondominant arm for local administration of study drugs. The catheter was connected to a pressure transducer for mean arterial pressure (MAP) measurement and was continuously flushed at a rate of 3 ml/h with heparinized saline (8). In five subjects (see protocol 2), an 18-gauge, 3-cm catheter was also inserted into an antecubital vein of the experimental forearm and directed toward the hand so the tip was located in a deep vein that drained the forearm muscles (19).
Forearm Blood Flow and Vascular Conductance
A 4-MHz pulsed Doppler probe (model 500V; Multigon Industries; Mt. Vernon, NY) was used to measure brachial artery mean blood velocity (MBV). The probe was securely fixed to the skin over the brachial artery proximal to the catheter insertion site as previously described by our laboratory (11, 50). The probe insonation angle was 60°. A linear 7.0-MHz echo Doppler ultrasound probe (128XP; Acuson; Mountain View, CA) was placed in a holder securely fixed to the skin immediately proximal to the velocity probe to measure brachial artery diameter. Forearm blood flow (FBF) was calculated as
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Rhythmic Handgrip Exercise
Rhythmic forearm handgrip exercise was performed using a 6.4-kg weight, which represents 10–15% of maximal voluntary contraction (MVC). The weight was lifted 4–5 cm over a pulley at a duty cycle of 1 s of contraction and 2 s of relaxation (20 contractions/min) using audio and visual signals to ensure correct timing.
Sympathetic -Adrenergic Vasoconstrictor Drugs
The following drugs were infused via the brachial artery catheter. Tyramine was infused at 8 µg/dl of forearm volume per minute to evoke endogenous norepinephrine release from sympathetic nerve endings (15) and subsequent postjunctional 1- and 2-adrenergic vasoconstriction (18). Importantly, tyramine does not have any direct vasoconstrictor effects (15), and the vascular responses to tyramine are abolished by nonselective -adrenergic blockade (9, 10). Because it is very difficult to assess endogenous norepinephrine release in response to tyramine under these experimental conditions, phenylephrine (a direct, selective 1-agonist) was infused at 0.03125 µg/dl of forearm volume per minute, and clonidine (a direct 2-agonist) was infused at 0.15 µg/dl of forearm volume per minute to determine postjunctional -adrenergic vasoconstrictor responsiveness. The doses of the vasoconstrictor drugs were based on recent findings that these doses cause significant vasoconstriction in the human forearm at rest (9) and during adenosine infusion and handgrip exercise (11, 38). All vasoconstrictor drug infusions were adjusted for hyperemic conditions.
To elevate resting FBF to levels similar to those observed during exercise, we infused adenosine (6.25 µg/dl of forearm volume per minute) via the brachial artery catheter ("passive" vasodilation). We previously demonstrated (11, 38, 50) that exercise blunts the vasoconstrictor responses to tyramine, phenylephrine, and clonidine, whereas these vasoconstrictor responses are maintained when blood flow is passively elevated with adenosine ("control" vasodilator condition). Therefore, sympathetic -adrenergic vasoconstrictor responses were compared during high-flow states in the presence (exercise) and absence (adenosine infusion) of muscle contractions. Importantly, all vasoconstrictor infusions were adjusted on the basis of steady-state FBF and forearm volume (measured via water displacement). These adjustments were made in an effort to normalize the concentrations of each constrictor drug in the blood perfusing the forearm across conditions where blood flow might differ. The concentrations of each compound were calculated to make sure the absolute infusion rate did not affect forearm hemodynamics (<3 ml/min in every trial).
Experimental Protocols
General experimental protocol.
Figure 1 is an example of a timeline for the specific trials. The subjects performed a bout of forearm exercise or received intra-arterial adenosine in a randomized manner; the total time for each trial was 9 min. After 2 min of baseline measurements, exercise or adenosine infusion was initiated and steady-state FBF was reached within 3 min. Between 3 and 4 min of hyperemia (minutes 5 and 6 on Fig. 1), the dose of the vasoconstricting agent was calculated on the basis of forearm volume and FBF. The vasoconstrictor infusion began at the 6-min mark and lasted for 3 min. A 15-min rest period was allowed between trials. The subjects who received all -adrenergic agonists underwent three trials of adenosine infusion and three trials of exercise before and after combined inhibition of NO and PGs (protocol 1) or inhibition of PGs alone (protocol 2).
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After these initial trials, NG-nitro-L-arginine methyl ester (L-NAME; Aerbio/Clinalfa) was administered at a rate of 5 mg/min for 10 min to inhibit NOS (total dose, 50 mg), and ketorolac (Toradol; Abbott Laboratories) was administered at a rate of 0.3 mg/min for 10 min to inhibit cyclooxygenase (COX; total dose, 3 mg). This dose of L-NAME reduces basal FBF as well as the vasodilator responses to acetylcholine, which is consistent with effective NOS blockade (11). Additionally, this dose of ketorolac causes a transient but consistent reduction in muscle blood flow when infused during steady-state handgrip exercise (41). Nevertheless, we have attempted to additionally demonstrate the efficacy of ketorolac to inhibit COX in protocol 2. Maintenance doses of L-NAME (1 mg/min) and ketorolac (0.3 mg/min) were continued throughout the rest of the experimental protocol to ensure continuous blockade of NOS and COX, respectively. Subsequent to combined NO and PG inhibition, the vasoconstrictor responses to the -adrenergic agonists were assessed again during adenosine infusion and handgrip exercise in randomized order. In all subjects, NO and PG inhibition was performed after the first set of adenosine infusion and exercise trials due to the long half-life of both inhibitors.
Protocol 2: Effects of PG inhibition alone on sympathetic vasoconstrictor responses during handgrip exercise.
Previous data indicate that NO inhibition alone does not affect functional sympatholysis in healthy humans (11). Therefore, this protocol was designed to determine the effects of PG inhibition alone on functional sympatholysis. In a group of six additional subjects, the vasoconstrictor responses to tyramine, phenylephrine, and clonidine were assessed during control vasodilator infusion of adenosine and during rhythmic handgrip exercise. Five of these subjects received all three vasoconstrictors during both hyperemic conditions, whereas one received only tyramine (due to time constraints of the subject). Thus the data presented for the tyramine trials reflect the average of all six subjects, and data presented for phenylephrine and clonidine reflect the average of five subjects. The infusion of each respective vasoconstrictor during adenosine infusion and handgrip exercise was randomized and counterbalanced across subjects, and the subjects rested for 15 min between each trial. After these initial trials, ketorolac (at the same loading and maintenance doses as for protocol 1) was given to inhibit COX. Subsequently, the vasoconstrictor responses to the -adrenergic agonists were assessed again during adenosine infusion and handgrip exercise in randomized order. In all subjects, COX inhibition was performed after the first set of adenosine infusion and exercise trials due to the long half-life of ketorolac (5).
In five of the subjects from protocol 2, the efficacy of our ketorolac dose to inhibit COX was determined by measuring arterial and deep venous concentrations of the stable metabolite of prostacyclin, 6-keto-prostaglandin F1 (PGF1), before and after COX inhibition using a commercially available chemiluminescence enzyme immunoassay kit (Assay Designs). The arterial and deep venous PGF1 concentrations were determined at baseline, and venous concentrations were also determined after 3 min of handgrip exercise for two randomly selected exercise trials both before and after ketorolac administration. The average values of arterial and venous concentrations of PGF1 from the two trials before ketorolac administration are presented and compared with the average of the two trials after PG inhibition.
Data Acquisition and Analysis
Data were collected and stored on computer at 250 Hz and were analyzed offline with signal-processing software (WinDaq; DATAQ Instruments; Akron, OH). MAP values were determined from the arterial pressure waveform. Baseline FBF and MAP values represent an average of the last minute of the resting time period, the hyperemic values represent an average of minutes 3–4 (minutes 5–6 of Fig. 1; pre-vasoconstrictor) during adenosine infusion or exercise, and the effects of the -agonists represent an average of the final 30 s of drug infusion (post-vasoconstrictor).
The percent reduction in FVC during vasoconstrictor administration was calculated as
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-agonists across conditions, as this has emerged as the most appropriate way to compare interventions that cause vasodilation or vasoconstriction under conditions where there might be marked differences in baseline blood flow (23, 31, 46, 50). However, in an effort to be comprehensive, we have presented the absolute levels of forearm hemodynamics during all conditions in tabular form. Statistics
All values are reported as means ± SE. Specific hypothesis testing within each of the exercise or adenosine administration trials with the three different drug infusions was performed using repeated-measures ANOVA. Comparisons of the hemodynamic values at specific time points between the exercise and adenosine conditions were made with unpaired t-tests, and the values within each hyperemic condition (exercise or adenosine infusion) before and after combined NO and PG inhibition (protocol 1) or before and after PG inhibition alone (protocol 2) were made with paired t-tests. Significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Forearm hemodynamics and MAP values for the experimental trials with each vasoconstrictor drug for protocol 1 are shown in Tables 1– 3. In general, adenosine infusion increased FBF and FVC values significantly, and the steady-state forearm hemodynamics were similar to those achieved during rhythmic handgrip exercise.
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FVC of –24 ± 4 vs. –63 ± 4%; P < 0.001; Fig. 2). Combined NO and PG inhibition reduced resting FVC by 50% as well as steady-state FVC during adenosine infusion (by 55%) and handgrip exercise (by 20%; Table 1). After combined inhibition, tyramine evoked similar vasoconstrictor responses during adenosine infusion (FVC, –57 ± 4%; P = 0.22), but the vasoconstrictor responses during exercise were augmented compared with the responses before NO and PG inhibition (FVC, –33 ± 3%; P < 0.01). However, the vasoconstriction observed during exercise was still significantly blunted compared with adenosine infusion (i.e., the responses were not completely restored; see Fig. 2).
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1-adrenergic receptor stimulation).
The vasoconstrictor responses to phenylephrine were significantly blunted during exercise compared with the responses during adenosine infusion (FVC, –10 ± 4 vs. –35 ± 3%; P < 0.001; Fig. 3). Combined NO and PG inhibition reduced resting FVC by 45% as well as steady-state FVC during adenosine (by 45%) and handgrip exercise (by 15%; Table 2). After combined inhibition, phenylephrine evoked vasoconstrictor responses that were augmented during exercise compared with before NO and PG inhibition (FVC, –20 ± 4%; P < 0.01). This was also observed for the vasoconstrictor responses during adenosine infusion (FVC, –45 ± 5%; P = 0.05). However, the vasoconstriction observed during exercise was still significantly blunted compared with adenosine infusion (i.e., the responses were not completely restored; see Fig. 3).
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2-adrenergic receptor stimulation).
Similar to tyramine and phenylephrine, the vasoconstrictor responses to clonidine were significantly blunted during exercise compared with the responses during adenosine infusion (FVC, –15 ± 3 vs. –49 ± 4%; P < 0.001; Fig. 4). Combined NO and PG inhibition reduced resting FVC by 50% as well as steady-state FVC during adenosine infusion (by 45%) and handgrip exercise (by 20%; Table 3). After combined blockade, clonidine evoked vasoconstrictor responses that were augmented during exercise compared with before NO and PG inhibition (FVC, –25 ± 3%; P < 0.01). This was also observed for the vasoconstrictor responses during adenosine infusion (FVC, –60 ± 3%; P = 0.05). However, the vasoconstriction observed during exercise was still significantly blunted compared with adenosine infusion (i.e., the responses were not completely restored; see Fig. 4).
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Forearm hemodynamics for the experimental trials with each vasoconstrictor drug for protocol 2 are shown in Table 4. Similar to protocol 1, adenosine infusion increased FBF and FVC values significantly, and the steady-state forearm hemodynamics were similar to those achieved during rhythmic handgrip exercise.
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-adrenergic agonists were significantly blunted during exercise compared with adenosine infusion (FVC: tyramine, –30 ± 4 vs. –61 ± 4%; phenylephrine, –13 ± 3 vs. –38 ± 5%; clonidine, –18 ± 4 vs. –41 ± 4%; all P < 0.05). PG inhibition alone did not significantly affect FBF, MAP (data not shown), or FVC values at rest or during the hyperemic conditions (all P > 0.05; Table 4). Additionally, PG inhibition alone did not influence the vasoconstrictor responses during adenosine infusion (FVC: tyramine, –66 ± 5; phenylephrine, –41 ± 8; clonidine, –43 ± 8%; P = 0.4–0.8) or during exercise (FVC: tyramine, –29 ± 6; phenylephrine, –12 ± 3; clonidine, –21 ± 3%; P = 0.6–0.9).
Before ketorolac administration, in five subjects, the baseline arterial concentration of PGF1 was 166 ± 23 pg/ml, the venous concentration was 225 ± 53 pg/ml, and the concentration during 3 min of handgrip exercise was 182 ± 33 pg/ml. After PG inhibition with ketorolac, arterial and deep venous concentrations of PGF1 at baseline were reduced to 124 ± 24 and 153 ± 13 pg/ml, respectively (26 and 32%, respectively), and the deep venous concentrations during exercise were reduced to 141 ± 23 pg/ml (23%; P = 0.04). Given that FBF values at baseline and during steady-state exercise were not different before and after ketorolac administration, these data are consistent with reduced prostacyclin production after COX inhibition with ketorolac.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-adrenergic vasoconstrictor responses in contracting human skeletal muscle. In contrast, PG inhibition alone does not influence sympathetic vasoconstriction during exercise. Thus taken together with our previous findings that NO is not obligatory for functional sympatholysis (11), it appears that inhibition of either NO or PGs alone does not affect functional sympatholysis in healthy humans, but combined inhibition can augment sympathetic vasoconstriction by 10%. However, the vasoconstrictor responses in active muscle after combined NO and PG inhibition are still substantially less than in resting muscle, which indicates that other signals must compensate under these conditions to preserve adequate blood flow and oxygen delivery to contracting muscle (51). Previous studies on contracting rat hindlimb have demonstrated a clear role for NO in functional sympatholysis (47, 48). However, recent findings from our laboratory indicate that NO is not obligatory for observation of this phenomenon in healthy humans (11), and this also appears to be the case for contracting dog hindlimb (7). These latter findings led us to believe that (similar to exercise hyperemia) there most likely is a redundancy in the factors involved in regulating functional sympatholysis, and the acute inhibition of one putative substance might be compensated for by an increased production of another substance and continue to blunt sympathetic vasoconstriction under these conditions.
Specifically, recent experimental data indicate that shear stress-induced prostacyclin production is significantly elevated during acute NOS inhibition, which suggests that PGs compensate for the lack of NO (32). Additionally, Sun et al. (43) demonstrated that flow-induced vasodilation is well preserved in endothelial NOS-knockout mice (compared with wild-type control mice), and this preserved vasodilator function is due to an increased contribution of vasodilating PGs. These data, when viewed in light of previous findings that PGs can inhibit norepinephrine release (25) as well as reduce postjunctional -adrenergic responsiveness (24), led us to hypothesize that an increased production of vasodilating PGs could possibly mask the role of NO in regulating functional sympatholysis when NO is inhibited.
To address this question, we determined sympathetic vasoconstrictor responses during adenosine infusion (control vasodilator) and rhythmic handgrip exercise before and after combined inhibition of NO and PGs (protocol 1). To the best of our knowledge, the present study is the first on humans to address whether combined inhibition of putative vasodilators involved in exercise hyperemia can restore sympathetic vasoconstriction in contracting muscle. Our findings indicate that combined inhibition of NO and PGs enhances the vasoconstrictor responses to direct postjunctional 1- and 2-adrenergic receptor stimulation during passive vasodilation (via adenosine) but not to endogenous norepinephrine release via tyramine. Whether this latter finding reflects different -receptor stimulation (abluminal receptors for tyramine vs. luminal receptors for direct agonists) or differences in norepinephrine release under these conditions is unknown. However, most importantly, we found that the vasoconstrictor responses to endogenous norepinephrine release and postjunctional 1- and 2-adrenergic stimulation are augmented in contracting muscle during combined inhibition of NO and PGs. Thus it appears that NO and PGs act together to partially blunt sympathetic -adrenergic vasoconstriction in contracting muscle.
Because we previously demonstrated that NO inhibition alone does not affect functional sympatholysis in healthy humans (11), we wanted to determine whether PG inhibition alone augments sympathetic vasoconstriction during exercise using the same experimental approach. In the present study, we found that PG inhibition alone (protocol 2) did not alter any of the vasoconstrictor responses in resting or contracting muscle, which is similar to what was demonstrated previously in rats (48) and humans (16). Taken together, these data indicate that acute inhibition of NO or PGs alone does not affect functional sympatholysis in healthy young humans, but combined inhibition of these substances can partially restore (by 10%) sympathetic vasoconstrictor responses in the vascular beds of contracting muscle.
Although our findings demonstrate that combined inhibition of NO and PGs can augment sympathetic vasoconstriction in contracting muscle, the responses are still significantly blunted compared with the responses under resting conditions. So, what other factors might be involved in functional sympatholysis in humans? Studies conducted by Thomas et al. (45) demonstrated that activation of ATP-sensitive potassium (KATP) channels during muscle contractions can attenuate sympathetic vasoconstriction in the rat hindlimb. Interestingly, in addition to NO and PGs, other factors that can activate these metabolically sensitive channels include adenosine, lactate, hydrogen ion, and tissue hypoxia (36). Therefore, it is possible that increased production of any of these "signals" during combined NO and PG inhibition continued to activate KATP channels and blunt sympathetic vasoconstriction. Another possibility that has yet to be tested is the role of endothelium-derived hyperpolarizing factor (EDHF), which acts in part via calcium-activated potassium (KCa) channels. EDHF appears to be especially important for maintaining endothelium-dependent vasodilation when NO and PGs are inhibited, and NO seems to have an inhibitory effect on EDHF production (3). Although the role of EDHF in modulating sympathetic vasoconstriction is not frequently studied, recent findings indicate that EDHF attenuates 1-mediated vasoconstriction (28) and evokes endothelium-dependent 2-mediated vasodilation (49), both of which could influence vasoconstrictor responses during exercise. To date, the roles of KATP and/or KCa channel activation in functional sympatholysis in humans have not been determined.
Experimental Considerations
One potential limitation to the present study is related to the effectiveness of L-NAME and ketorolac to completely inhibit NOS and COX, respectfully. Recently, we demonstrated that the same dose of L-NAME used in this study reduces resting vascular tone and the vasodilator responses to acetylcholine (11), which is consistent with effective NOS blockade. Additionally, the dose we used is substantially greater (on a per-kilogram basis) than a systemic dose that was shown to reduce NOS activity by 70% (14). With respect to COX inhibition, the dose of ketorolac administered in our study is approximately sevenfold greater (on a per-kilogram basis) than what is clinically used as an analgesic for moderate to severe acute pain (5). Although this did not reduce FBF or FVC values at rest (as was demonstrated in some studies using indomethacin to inhibit COX; Refs. 12, 53), this is consistent with other studies that have clearly shown effects of PG inhibition on acetylcholine-mediated vasodilator responses without any changes in resting vascular tone (27, 33, 44). Additionally, in a subset of subjects, we demonstrated that this dose of ketorolac reduced arterial and deep forearm venous concentrations of the stable metabolite of prostacyclin, 6-keto-PGF1. Finally, in a recent study in our laboratory designed to understand the roles of NO and PGs in regulating exercise hyperemia, we found that this dose of ketorolac transiently but consistently reduced FBF by 12% when infused during steady-state handgrip exercise (41). Taken together, we believe that we gave adequate doses of L-NAME and ketorolac to inhibit NO and PG synthesis, respectfully, in the forearm vasculature.
Another potential limitation relates to the significant reduction in steady-state forearm hemodynamic responses to adenosine during combined NO and PG inhibition and, in turn, the comparison of sympathetic vasoconstrictor responses with those observed during the other hyperemic conditions. Although this represents a significant difference in "baseline" hemodynamics from which vasoconstrictor responses are calculated, there is substantial evidence to indicate that use of percentage changes in vascular conductance (as used in the present study) is clearly the most accurate way to quantify vasoconstrictor responses under these conditions (23, 31, 46, 50). Furthermore, all of the vasoconstrictor infusions were adjusted to the steady-state level of FBF during each condition to keep the concentration of the vasoconstricting substance in the blood perfusing the forearm similar across conditions. Therefore, despite differences in forearm hemodynamics in response to adenosine infusion during combined NO and PG inhibition, we believe that we are appropriately quantifying the vasoconstrictor responses to the same vasoconstrictor stimuli across conditions, and this should not limit the interpretation of our data. Finally, the key finding from the present study was that combined NO and PG inhibition augments sympathetic -adrenergic vasoconstrictor responses during exercise (when there was not much effect on steady-state forearm hemodynamics).
Boushel et al. (4) recently demonstrated that combined inhibition of NO and PGs reduces muscle blood flow during knee extensor exercise in humans, a finding that somewhat contrasts with what we observed in the present study during handgrip exercise (see Tables 1–3). However, as recently discussed (41), combined NO and PG inhibition did not significantly influence quadriceps muscle blood flow at low workloads during exercise. Therefore, our results during mild forearm handgrip exercise are consistent with this previous study during low-intensity knee extensor exercise. Interestingly, in this study by Boushel et al. (4), the magnitude of the reduction in muscle blood flow was more pronounced as exercise intensity and duration increased. At these workloads and exercise durations using a larger muscle mass, it is quite possible that sympathetic vasoconstrictor nerve activity was progressively increased (17, 42), and this idea is supported by the progressive increase in blood pressure during exercise (4). Therefore, when viewed in the context of the findings from the present study, it is not entirely clear how much of the reduction in muscle blood flow during combined NO and PG inhibition in their study was due to the removal of the vasodilator properties of these substances, or whether this resulted in the partial restoration of sympathetic vasoconstriction in active muscle. Future studies will be needed to address this issue.
Potential Implications
Exercise tolerance is reduced with age and in patients with congestive heart failure in part due to reductions in muscle blood flow during exercise (22, 34). Although this might reflect an impairment of the skeletal muscle vasculature to vasodilate in response to various metabolic or flow-induced signals, this might also reflect an impaired ability to blunt sympathetic vasoconstriction during large-muscle dynamic exercise (21). In both populations, endothelial function is significantly impaired, and this is reflected by a reduction in NO bioavailability and oxidative stress-induced production of vasoconstrictor prostanoids (44, 52). Additionally, sympathetic activity is elevated in these populations compared with healthy control subjects at rest and during exercise (22, 35). Taken together, it seems plausible to speculate that a reduction in NO and vasodilating PGs in healthy older adults and heart failure patients could impair the ability of muscle contractions to blunt sympathetic vasoconstriction and thereby result in corresponding reductions in muscle blood flow and exercise intolerance.
In summary, the findings from the present investigation demonstrate that combined inhibition of NO and PGs augments sympathetic -adrenergic vasoconstriction in contracting skeletal muscle of humans. When viewed in the context of recent findings from our laboratory, it appears that inhibition of NO or PGs alone does not affect functional sympatholysis, which suggests that there is some compensation by these substances when the other substance is inhibited. However, the vasoconstrictor responses in exercising muscle during combined NO and PG inhibition were still substantially blunted compared with those at rest, which indicates that other signals must compensate under these conditions to preserve adequate blood flow and oxygen delivery to contracting muscle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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B. S. Kirby, W. F. Voyles, R. E. Carlson, and F. A. Dinenno Graded sympatholytic effect of exogenous ATP on postjunctional {alpha}-adrenergic vasoconstriction in the human forearm: implications for vascular control in contracting muscle J. Physiol., September 1, 2008; 586(17): 4305 - 4316. [Abstract] [Full Text] [PDF]
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B. S. Kirby, R. E. Carlson, R. R. Markwald, W. F. Voyles, and F. A. Dinenno Mechanical influences on skeletal muscle vascular tone in humans: insight into contraction-induced rapid vasodilatation J. Physiol., September 15, 2007; 583(3): 861 - 874. [Abstract] [Full Text] [PDF]
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B. W. Wilkins, W. G. Schrage, Z. Liu, K. C. Hancock, and M. J. Joyner Systemic hypoxia and vasoconstrictor responsiveness in exercising human muscle J Appl Physiol, November 1, 2006; 101(5): 1343 - 1350. [Abstract] [Full Text] [PDF]
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 W. Zhao, S. A. Swanson, J. Ye, X. Li, J. M. Shelton, W. Zhang, and G. D. Thomas Reactive Oxygen Species Impair Sympathetic Vasoregulation in Skeletal Muscle in Angiotensin II-Dependent Hypertension Hypertension, October 1, 2006; 48(4): 637 - 643. [Abstract] [Full Text] [PDF]
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N. Charkoudian, M. J. Joyner, S. A. Barnes, C. P. Johnson, J. H. Eisenach, N. M. Dietz, and B. G. Wallin Relationship between muscle sympathetic nerve activity and systemic hemodynamics during nitric oxide synthase inhibition in humans Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1378 - H1383. [Abstract] [Full Text] [PDF]
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B. S. Kirby, R. R. Markwald, E. G. Smith, and F. A. Dinenno Mechanical effects of muscle contraction do not blunt sympathetic vasoconstriction in humans Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1610 - H1617. [Abstract] [Full Text] [PDF]
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F. A Dinenno, S. Masuki, and M. J Joyner Impaired modulation of sympathetic {alpha}-adrenergic vasoconstriction in contracting forearm muscle of ageing men J. Physiol., August 15, 2005; 567(1): 311 - 321. [Abstract] [Full Text] [PDF]
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L. A Holowatz, C. S Thompson, C. T Minson, and W. L. Kenney Mechanisms of acetylcholine-mediated vasodilatation in young and aged human skin J. Physiol., March 15, 2005; 563(3): 965 - 973. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. exercise before L-NAME and ketorolac infusions.
