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. 1998 Feb 1;506(Pt 3):817–826. doi: 10.1111/j.1469-7793.1998.817bv.x

Nitric oxide mediates contraction-induced attenuation of sympathetic vasoconstriction in rat skeletal muscle

Gail D Thomas 1, Ronald G Victor 1
PMCID: PMC2230749  PMID: 9503340

Abstract

  1. Sympathetic vasoconstriction is attenuated by metabolic events in contracting rat skeletal muscle, in part by activation of ATP-sensitive potassium (KATP) channels. However, the specific metabolites in contracting muscle that open KATP channels are not known. We therefore asked if contraction-induced attenuation of sympathetic vasoconstriction is mediated by the endogenous vasodilators nitric oxide (NO), adenosine, or prostaglandins PGI2 or PGE2, all of which are putative KATP channel openers.

  2. In anaesthetized rats, hindlimb contraction alone significantly attenuated the vasoconstrictor responses to lumbar sympathetic nerve stimulation. Inhibition of NO synthase with N-nitro-L-arginine methyl ester (L-NAME, 5 mg kg−1, i.v.) partially reversed this effect of contraction, resulting in enhanced sympathetic vasoconstriction in contracting hindlimb. Subsequent treatment with the KATP channel blocker glibenclamide (20 mg kg−1, i.v.) had no further effect on sympathetic vasoconstriction in contracting hindlimb.

  3. This effect of L-NAME to partially reverse contraction-induced attenuation of sympathetic vasoconstriction was not replicated by D-NAME (5 mg kg−1, i.v.) or angiotensin II (12.5 ng kg−1 min−1, i.v.), the latter used as a hypertensive control.

  4. Adenosine receptor blockade with 8-(p-sulphophenyl)theophylline (10 mg kg−1, i.v.) or cyclooxygenase inhibition with indomethacin (5 mg kg−1, i.v.) had no effect on contraction-induced attenuation of sympathetic vasoconstriction.

  5. These results suggest that NO plays an important role in the precise regulation of blood flow in exercising skeletal muscles by opposing sympathetic vasoconstriction. Although the underlying mechanism is not known, it may involve NO-induced activation of vascular KATP channels.

Skeletal muscle contraction activates chemically sensitive muscle afferents which are thought to signal the brain of a mismatch between muscle perfusion and metabolic need and elicit reflex increases in efferent sympathetic nerve discharge. Direct recordings of muscle sympathetic nerve activity have shown that this exercise-induced reflex increase in sympathetic discharge is targeted to both resting and exercising skeletal muscle (Hansen, Thomas, Jacobsen & Victor, 1994). In quiescent skeletal muscle, sympathetic activation produces vasoconstriction, which redistributes blood flow to the exercising muscles, thereby optimizing muscle perfusion. In contracting skeletal muscle, however, the functional consequence of sympathetic activation has been more difficult to define.

Previous studies in intact animals and humans have provided evidence to suggest that the normal ability of sympathetic nerve activation to produce vasoconstriction can be greatly attenuated by muscle contraction (Remensnyder, Mitchell & Sarnoff, 1962; Strandell & Shepherd, 1967; Rowlands & Donald, 1968; Burcher & Garlick, 1973). Further support for this concept, initially termed functional sympatholysis (Remensnyder et al. 1962), has been generated from experiments in reductionist microvascular preparations demonstrating that certain local metabolic consequences of contraction (e.g. acidosis, hypoxia) inhibit α-adrenergic vasoconstriction (McGillivray-Anderson & Faber, 1990, 1991; Anderson & Faber, 1991). Recent studies from our laboratory in anaesthetized rats and conscious humans demonstrated that functional sympatholysis negates an otherwise deleterious effect of sympathetic activation on skeletal muscle perfusion and oxygenation (Thomas, Hansen & Victor, 1994; Hansen, Thomas, Harris, Parsons & Victor, 1996). However, the underlying metabolic conditions in contracting muscle that attenuate sympathetic vasoconstriction are incompletely understood.

In quiescent skeletal muscle, sympathetic vasoconstriction is mediated by activation of both postjunctional α1- and α2-adrenergic receptors (Ohyanagi, Faber & Nishigaki, 1991). α2-mediated vasoconstriction in particular is very sensitive to inhibition by increased tissue metabolism, and is preferentially attenuated by skeletal muscle contraction (Anderson & Faber, 1991; Thomas et al. 1994). The underlying basis for this metabolic sensitivity of α2- receptors is not known, but it may be mediated in part by interaction with a specific class of metabolically regulated vascular potassium channels, the ATP-dependent potassium (KATP) channels. Opening of these channels has been shown to relax vascular smooth muscle and attenuate α-adrenergic vasoconstriction, with a greater inhibitory effect on α2-, rather than α1-mediated vasoconstriction (Mori et al. 1995; Tateishi & Faber, 1995). We recently demonstrated that this interaction between KATP channel activation and α2-adrenergic vasoconstriction is one important mechanism underlying functional sympatholysis in rat hindlimb (Thomas, Hansen & Victor, 1997). However, the specific metabolic events in contracting muscle that open KATP channels are not known.

Contracting skeletal muscle produces a number of vasodilator metabolites that have been postulated to act, at least in part, by opening KATP channels (Quayle & Standen, 1994). These include nitric oxide (NO), adenosine and prostaglandins PGI2 and PGE2. All these substances are potent vasodilators which have been implicated in the regulation of local blood flow in quiescent, as well as contracting, skeletal muscle (Green, O'Driscoll, Blanksby & Taylor, 1996). In addition, each of these metabolites has been reported to attenuate α-adrenergic vasoconstriction (Gottlieb, Lippton, Parey, Paustian & Kadowitz, 1980; Nishigaki, Faber & Ohyanagi, 1991; Ohyanagi, Nishigaki & Faber, 1992; Patil, DiCarlo & Collins, 1993), suggesting that one or more of these substances could be the endogenous mediator underlying functional sympatholysis.

The aims of the present study, therefore, were twofold. First, using our anaesthetized rat model, we wanted to determine the relative contributions of NO, adenosine, and the vasodilatory prostaglandins in mediating contraction-induced attenuation of α-adrenergic vasoconstriction. Second, we wanted to determine if the underlying mechanism by which any of these metabolites attenuates vasoconstriction involves the activation of KATP channels.

METHODS

Experimental preparation

All the surgical and experimental protocols used in this study were approved by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center.

Thirty-nine female Sprague-Dawley rats (206-304 g; Charles River, Kingston, MA, USA) were anaesthetized with ketamine (80 mg kg−1, i.p.) and α-chloralose (60 mg kg−1, i.v., followed by 10 mg kg−1, i.v. per hour). Atropine sulphate (0.5 mg kg−1, s.c.) was administered and a jugular vein and carotid artery were cannulated. The latter was used to measure arterial pressure. The cervical trachea was cannulated and the animals were ventilated with room air and supplemental oxygen. Arterial blood gases were measured periodically (ABL-3, Radiometer, Copenhagen, Denmark) and kept within normal limits. Core temperature was maintained at 37°C with an external heat source.

A Doppler flow probe was placed around the left femoral artery to measure changes in blood flow velocity by recording the pulsatile and mean Doppler shifts in kHz using a VF-1 pulsed Doppler flow system (Crystal Biotech, Holliston, MA, USA). Femoral vascular conductance (kHz mmHg−1) was calculated as the mean Doppler shift divided by mean arterial pressure.

The left lumbar sympathetic chain was exposed via an anterior abdominal incision, isolated inferior to the renal artery, placed on bipolar platinum electrodes and covered with silicone rubber (SilGel 604, Wacker-Chemie, Munich, Germany). The lumbar nerve was electrically stimulated (model S88, Grass Instruments, Quincy, MA, USA) for 1 min at 2.5 or 5 Hz with 1 ms pulses of 5 V. In some experiments, a small non-occlusive catheter was placed in the abdominal aorta where it was situated proximal to the iliac bifurcation and was used for intra-arterial hindlimb infusion of the α2-adrenergic agonist UK 14304.

The left sciatic nerve was exposed in the region of the sciatic notch, covered with warm mineral oil, and affixed to stimulating electrodes. The left triceps surae muscles were dissected free of surrounding muscles and connected to a force-displacement transducer (FT-10, Grass Instruments) via the calcaneal tendon. To produce intermittent, tetanic contractions, the sciatic nerve was stimulated (model S88, Grass Instruments) at 2-3 times the motor threshold voltage with 100 ms trains of pulses (100 Hz, 0.2 ms duration) at a rate of 60 trains min−1. Contraction periods of 10-20 min were separated by rest periods of at least 20 min. At the end of the experiments, animals were killed with an overdose of sodium pentobarbitone (150 mg kg−1, i.v.).

Drugs

Nω-Nitro-L-arginine methyl ester (L-NAME), N ω-nitro-D-arginine methyl ester (D-NAME), and [β-Asp1]-angiotensin II (Sigma) were dissolved in 0.9 % saline. UK 14304 (RBI, Natick, MA, USA) was dissolved initially in dimethylsulphoxide (DMSO) to make a 10 mg ml−1 stock solution and then further diluted to 10 μg ml−1 in 0.9 % saline. 8-(p-Sulphophenyl)theophylline (8-SPT) and 2-chloroadenosine (both from RBI) were dissolved in water. Indomethacin (RBI) was dissolved in 5 % NaOH. Glibenclamide (RBI) was dissolved in 0.1 M NaOH under continuous sonication at a concentration of 25 mg ml−1 and then was diluted to 5 mg ml−1 in 5 % dextrose solution.

Experimental protocols

Protocol 1. Effect of pharmacological inhibition of nitric oxide synthase (NOS) on the contraction-induced attenuation of sympathetic vasoconstriction (n= 27 rats)

The purpose of this protocol was to determine if NOS inhibition would restore the vasoconstrictor responses to α-adrenergic stimulation in contracting skeletal muscle. We measured the mean arterial pressure and femoral blood flow velocity responses to lumbar sympathetic nerve stimulation (n= 10) or intra-arterial hindlimb infusion of the α2-adrenergic agonist UK 14304 (n= 7) in resting and contracting hindlimb before and after NOS inhibition with L-NAME (5 mg kg−1, i.v.). In a subset (n= 5) of the former group of animals (with lumbar nerve stimulation), a third contraction period was performed after KATP channel blockade with glibenclamide (20 mg kg−1, i.v.). In all the experiments, sympathetic nerve stimulation or intra-arterial infusion of UK 14304 was performed when the force produced by the contracting muscles had declined to ∼ 50 % of the initial value. Separate control experiments were performed using D-NAME (5 mg kg−1, i.v.; n= 5), the inactive enantiomer of L-NAME, or angiotensin II (12.5 ng kg−1 min−1, i.v.; n= 5), used to mimic the hypertensive effect of L-NAME.

Protocol 2. Effect of pharmacological blockade of adenosine receptors on the contraction-induced attenuation of sympathetic vasoconstriction (n= 6 rats)

To determine if adenosine produced by contracting skeletal muscle is responsible for the attenuated sympathetic vasoconstriction, we measured the mean arterial pressure and femoral blood flow velocity responses to lumbar sympathetic nerve stimulation in resting and contracting hindlimb before and after adenosine receptor blockade with 8-SPT (10 mg kg−1, i.v.; n= 6). In five of these animals, a third contraction period was performed after KATP channel blockade with glibenclamide (20 mg kg−1, i.v.).

Protocol 3. Effect of pharmacological inhibition of cyclooxygenase on the contraction-induced attenuation of sympathetic vasoconstriction (n= 6 rats)

To determine if prostaglandins contribute to the contraction-induced attenuation of sympathetic vasoconstriction, we measured the mean arterial pressure and femoral blood flow velocity responses to lumbar sympathetic nerve stimulation in resting and contracting hindlimb before and after cyclo-oxygenase inhibition with indomethacin (5 mg kg−1, i.v.; n= 6). In five of these animals, a third contraction period was performed after KATP channel blockade with glibenclamide (20 mg kg−1, i.v.).

Statistics

Statistical analysis was performed using one-way or two-way repeated measures analysis of variance with Scheffe's post hoc test. Differences were considered statistically significant when P < 0.05. Results are expressed as means ±s.e.m.

RESULTS

Pharmacological inhibition of NOS partially restores the vasoconstrictor response to sympathetic nerve stimulation in contracting hindlimb

Before L-NAME, lumbar sympathetic nerve stimulation elicited significant decreases in femoral blood flow and vascular conductance in resting hindlimb (Figs 1 and 2). As expected, these vasoconstrictor responses were significantly attenuated by hindlimb contraction, indicating functional sympatholysis (Figs 1 and 2).

Figure 1. Effects of L-NAME on femoral blood flow responses to sympathetic stimulation in resting and contracting hindlimb.

Figure 1

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Segments of an original record from one rat showing the arterial pressure and femoral blood flow velocity responses to lumbar sympathetic nerve stimulation (Symp. Stim.) in resting and contracting hindlimb before (top) and after (bottom) NOS inhibition with L-NAME. Before L-NAME, sympathetic stimulation decreased femoral blood flow in resting muscle. This vasoconstrictor effect was greatly attenuated during hindlimb contraction (i.e. functional sympatholysis) as indicated by the increase, rather than decrease, in femoral blood flow velocity elicited by sympathetic nerve stimulation. In this same rat after L-NAME, sympathetic nerve stimulation elicited a decrease in femoral blood flow velocity, indicating that sympathetic vasoconstriction was partially restored in the contracting muscles.

Figure 2. Effects of L-NAME and subsequent administration of glibenclamide (GB) on the femoral vascular conductance responses to sympathetic stimulation in resting and contracting hindlimb.

Figure 2

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Summary data showing the decreases in femoral vascular conductance in response to lumbar sympathetic nerve stimulation in resting (top) and contracting (bottom) hindlimb in the absence of any drug (n= 10), and after infusion of L-NAME alone (n= 10) or in combination with GB (n= 5). Sympathetic stimulation produced robust vasoconstrictor responses in resting hindlimb that were not affected by either of the drugs. Hindlimb contraction significantly attenuated these vasoconstrictor responses in the absence of any drug. L-NAME partially reversed this attenuation, so that sympathetic stimulation elicited a significantly greater vasoconstrictor response in contracting hindlimb. This effect of L-NAME in contracting hindlimb was not further enhanced by the addition of GB.

Pretreatment with L-NAME significantly increased mean arterial pressure (+45 ± 4 mmHg) and decreased femoral vascular conductance in resting hindlimb (Table 1). Despite these baseline effects, L-NAME had no significant effect on the decreases in femoral blood flow and vascular conductance in response to lumbar nerve stimulation in resting hindlimb (Fig. 2). L-NAME also had no significant effect on the hyperaemic response to hindlimb contraction alone (Table 1) or on the force produced by the contracting muscles (peak force, 1.9 ± 0.1 kg before vs. 1.9 ± 0.1 kg after, L-NAME). In contrast, L-NAME significantly enhanced the vasoconstrictor responses to lumbar sympathetic nerve stimulation in contracting hindlimb (Figs 1 and 2).

Table 1.

Comparative effects of L-NAME, D-NAME and angiotensin II in the haemodynamic responses to hindlimb contraction

Before drug After drug
Group Rest Contraction Δ Rest Contraction Δ
Mean arterial pressure (mmHg)
L-NAME 77 ± 3 80 ± 4 +4 ± 5 126 ± 5 * 125 ± 5 * −1 ± 5
D-NAME 78 ± 2 86 ± 7 +8 ± 6 78 ± 5 92 ± 6 +14 ± 5
Angiotensin II 71 ± 3 84 ± 3 +12 ± 3 112 ± 2 * 123 ± 2 * +10 ± 1
Femoral blood flow velocity (kHz)
L-NAME 0.94 ± 0.11 3.12 ± 0.33 +2.18 ± 0.24 1.09 ± 0.14 4.10 ± 0.61 * +3.00 ± 0.49 *
D-NAME 0.92 ± 0.13 4.11 ± 0.49 +3.19 ± 0.38 1.13 ± 0.20 4 30 ± 0.68 +3.17 ± 0.54
Angiotensin II 0.83 ± 0.13 3.04 ± 0.71 +2.21 ± 0.64 1.55 ± 0.15 * 4.51 ± 0.98 * +2.96 ± 0.83
Femoral vascular conductance (kHz mmHg−1)
L-NAME 0.012 ± 0.001 0.039 ± 0.004 +0.027 ± 0.003 0.009 ± 0.001 * 0.033 ± 0.005 * +0.024 ± 0.004
D-NAME 0.012 ± 0.001 0.047 ± 0.003 +0.036 ± 0.001 0.014 ± 0.002 0.046 ± 0.006 +0.032 ± 0.004
Angiotensin II 0.012 ± 0.002 0.037 ± 0.009 +0.025 ± 0.009 0.014 ± 0.001 0.037 ± 0.008 +0.023 ± 0.007
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Values are means ±s.e.m. L-NAME (5 mg kg−1, i.v.), n= 17; D-NAME (5 mg kg−1, i.v.), n = 5; angiotensin II (12.5 ng kg−1 min−1), n= 5.

*

P< 0.05 vs. Before drug.

Subsequent treatment with glibenclamide had no further effect on the L-NAME-induced increase in arterial pressure, but it did significantly decrease femoral blood flow and vascular conductance in the hindlimb at rest (-33 ± 6 and -38 ± 6 %, respectively), as well as during contraction (-38 ± 8 and -42 ± 8 %, respectively). Glibenclamide did not affect sympathetic vasoconstriction in resting hindlimb, nor did it further potentiate the effect of L-NAME alone, to enhance sympathetic vasoconstriction in contracting hindlimb (Fig. 2). We previously have reported that glibenclamide alone partially reverses functional sympatholysis in this rat model (Thomas et al. 1997).

Pharmacological inhibition of NOS also partially restores the vasoconstrictor response to infusion of an α2-adrenergic agonist in contracting hindlimb

Before L-NAME, intra-arterial infusion of the α2-adrenergic agonist UK 14304 decreased femoral blood flow (-54 ± 3 %) and vascular conductance (-63 ± 2 %) in resting hindlimb. These α2-mediated vasoconstrictor responses were significantly attenuated by hindlimb contraction alone (flow, +37 ± 4 %; conductance, +4 ± 2 %; P < 0.05vs. responses in resting hindlimb). L-NAME had no effect on UK 14304-induced vasoconstriction in resting hindlimb. However, L-NAME did significantly enhance the vasoconstrictor responses to UK 14304 during hindlimb contraction (blood flow, -8 ± 5 %; conductance, -18 ± 2 %; P < 0.05vs. responses in contracting muscle before L-NAME).

In contrast to L-NAME, neither D-NAME nor angiotensin II restores sympathetic vasoconstriction in contracting hindlimb

The haemodynamic responses to hindlimb contraction alone, the vasoconstrictor responses to sympathetic nerve stimulation in resting hindlimb, and the effect of hindlimb contraction to attenuate sympathetic vasoconstriction were not significantly altered by either D-NAME or angiotensin II, the latter used to increase arterial pressure by 41 ± 3 mmHg to mimic the hypertensive effect of L-NAME (Table 1; Fig. 3).

Figure 3. Effects of D-NAME or angiotensin II (Ang II) on the femoral vascular conductance responses to sympathetic stimulation in resting and contracting hindlimb.

Figure 3

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Summary data showing the decreases in femoral vascular conductance in response to lumbar sympathetic nerve stimulation in resting (top) and contracting (bottom) hindlimb before and after D-NAME (n= 5; left panels), or Ang II (n= 5; right panels). Sympathetic stimulation produced decreases in conductance in resting hindlimb that were not affected by either of the drugs. Hindlimb contraction significantly attenuated these vasoconstrictor responses in the absence of either drug. This contraction-induced attenuation of sympathetic vasoconstriction was not affected by either D-NAME or Ang II (used to increase arterial pressure by 41 ± 3 mmHg).

Neither adenosine receptor blockade nor cyclooxygenase inhibition restores sympathetic vasoconstriction in contracting hindlimb

Before 8-SPT or indomethacin, lumbar sympathetic nerve stimulation elicited significant decreases in femoral blood flow and vascular conductance in resting hindlimb (Fig. 4). As expected, these vasoconstrictor responses were significantly attenuated by hindlimb contraction alone (Fig. 4).

Figure 4. Effects of 8-SPT or indomethacin (Indo) alone and in combination with glibenclamide (GB) on the femoral vascular conductance responses to sympathetic stimulation in resting and contracting hindlimb.

Figure 4

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Summary data showing the decreases in femoral vascular conductance in response to lumbar sympathetic nerve stimulation in resting (top) and contracting (bottom) hindlimb before and after 8-SPT alone (n= 6) and in combination with GB (n= 5; left panels), or Indo alone (n= 6) and in combination with GB (n= 5) (right panels). Sympathetic stimulation produced robust vasoconstrictor responses in resting hindlimb that were not affected by any of the drugs. Hindlimb contraction alone significantly attenuated these vasoconstrictor responses. This contraction-induced attenuation of sympathetic vasoconstriction was not affected by either 8-SPT or Indo. However, the addition of GB partially reversed this attenuation in both 8-SPT-treated and Indo-treated rats, as evidenced by the significantly greater vasoconstrictor responses to sympathetic stimulation in contracting hindlimb after GB.

Neither 8-SPT nor indomethacin significantly affected the vasoconstrictor responses to sympathetic nerve stimulation in resting hindlimb (Fig. 4). Also, neither drug significantly altered the effect of hindlimb contraction to attenuate sympathetic vasoconstriction (Fig. 4). Neither the hyperaemic response to hindlimb contraction alone (Table 2), nor the peak forces produced by the contracting muscles was affected by 8-SPT (1.7 ± 0.1 kg before vs. 1.7 ± 0.1 kg after) or by indomethacin (1.7 ± 0.1 kg before vs. 1.6 ± 0.1 kg after). In the former experiments, the efficacy of 8-SPT to block adenosine receptors was determined by measuring the peak hypotensive response to 2-chloroadenosine (30 μg kg−1, i.v.) (-19 ± 2 mmHg before vs. -5 ± 3 mmHg after 8-SPT; P < 0.05).

Table 2.

Comparative effects of 8-SPT and indomethacin on the haemodynamic responses to hindlimb contraction

Before drug After drug
Group Rest Contraction Δ Rest Contraction Δ
Mean arterial pressure (mmHg)
8-SPT 68 ± 1 73 ± 4 +5 ± 3 71 ± 3 78 ± 5 +7 ± 2
Indomethacin 90 ± 7 100 ± 5 +10 ± 5 85 ± 7 97 ± 5 +12 ± 4
Femoral blood flow velocity (kHz)
8-SPT 1.10 ± 0.10 3.20 ± 0.27 +2.11 ± 0.21 1.83 ± 0.26 * 3.45 ± 0.37 +1.63 ± 0.24
Indomethacin 0.89 ± 0.05 3.75 ± 0.45 +2.90 ± 0.40 1.90 ± 0.13 4.04 ± 0.45 +2.94 ± 0.37
Femoral vascular conductance (kHz mmHg−1)
8-SPT 0.016 ± 0.002 0.045 ± 0.005 +0.029 ± 0.004 0.026 ± 0.004 * 0.045 ± 0.004 +0.019 ± 0.002
Indomethacin 0.010 ± 0.001 0.038 ± 0.004 +0.028 ± 0.004 0.013 ± 0.001 * 0.042 ± 0.005 +0.029 ± 0.005
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Values are means ±s.e.m. 8-SPT (10 mg kg−1, i.v.), n = 6; indomethacin (5 mg kg−1, i.v.), n = 6.

*

P < 0.05vs. Before drug.

Subsequent treatment with glibenclamide significantly increased mean arterial pressure in both the 8-SPT-treated (+10 ± 3 mmHg) and indomethacin-treated (+10 ± 6 mmHg) rats. In resting hindlimb, sympathetic vasoconstriction was not affected by glibenclamide in either group of rats (Fig. 4). The increase in femoral vascular conductance in response to hindlimb contraction alone was significantly attenuated by glibenclamide in both groups of rats (8-SPT, -28 ± 7 %; indomethacin, -36 ± 5 %). In contrast to the lack of an effect of 8-SPT or indomethacin alone on contraction-induced attenuation of sympathetic vasoconstriction, glibenclamide partially reversed functional sympatholysis in both groups of rats (Fig. 4). These effects of glibenclamide given after 8-SPT or indomethacin are similar to the effects of glibenclamide alone, as we have reported previously in this rat model (Thomas et al. 1997).

DISCUSSION

Studies from our laboratory in anaesthetized rats and conscious humans have demonstrated that contraction-induced attenuation of sympathetic vasoconstriction (i.e. functional sympatholysis) negates an otherwise deleterious effect of sympathetic activation on skeletal muscle blood flow and oxygenation (Thomas et al. 1994; Hansen et al. 1996). We recently demonstrated that activation of KATP channels is one important mechanism underlying functional sympatholysis (Thomas et al. 1994). We now implicate a major role for NO, but not adenosine or prostaglandins, in mediating functional sympatholysis. Our data further suggest that one mechanism by which NO attenuates sympathetic vasoconstriction may involve an interaction with KATP channels.

NO is a particularly attractive candidate for mediating functional sympatholysis for a number of reasons. First, current evidence suggests that NO production increases during skeletal muscle contraction (Balon & Nadler, 1994; Node et al. 1997). Second, NO has been shown to antagonize α-adrenergic vasoconstriction in isolated blood vessels (Martin, Furchgott, Villani & Jothianidine, 1986; Topouzis, Schott & Stoclet, 1991), in the skeletal muscle microcirculation of anaesthetized rats (Ohyanagi et al. 1992), and in the non-exercising hindlimb of conscious rats (Patil et al. 1993). Third, this antagonistic effect of NO is more potent for α2-, rather than α1-adrenergic vasoconstriction in the rat skeletal muscle microcirculation (Ohyanagi et al. 1992) and canine coronary artery (Jones, DeFily, Patterson & Chilian, 1993). A similar preferential sensitivity of α2-mediated vasoconstriction to metabolic inhibition is one of the defining characteristics of functional sympatholysis in intact rats (Thomas et al. 1994). Thus, our finding that NO mediates functional sympatholysis is entirely consistent with a greater sensitivity of α2-adrenergic vasoconstriction to inhibition by NO.

To the best of our knowledge, this study is the first to demonstrate that NO attenuates α-adrenergic vasoconstriction in exercising skeletal muscle. This effect of NO may persist in the early post-exercise period, as suggested by a previous study in which NOS inhibition with L-NAME potentiated α-adrenergic vasoconstriction in the hindlimb of conscious rats after exhaustive treadmill exercise (Patil et al. 1993). In contrast, when blood flow was measured during intermittent interruptions of rhythmic forearm exercise in humans, NOS inhibition with N-monomethyl-L-arginine (L-NMMA) did not potentiate noradrenaline-induced forearm vasoconstriction (Wilson & Kapoor, 1993). In preliminary experiments, we found that although L-NMMA appeared to enhance sympathetic vasoconstriction in contracting rat hindlimb, it was less effective than L-NAME.

An alternative explanation for the effect of L-NAME to restore sympathetic vasoconstriction in contracting rat skeletal muscle might be activation of local myogenic reflexes attendant to the L-NAME-induced elevation in arterial pressure. However, this explanation is unlikely because sympathetic vasoconstriction was not restored in contracting hindlimb when we mimicked the L-NAME-induced increase in arterial pressure with angiotensin II. We further established that the effect of L-NAME to partially reverse functional sympatholysis was specific for NOS inhibition by demonstrating that D-NAME, the inactive enantiomer of L-NAME, had no effect on sympatholysis.

The source of the NO that interacts with sympathetic vasoconstriction in contracting skeletal muscle and the stimulus for its release are unknown. NO could be derived either from the endothelial isoform of NOS located in the vascular endothelium, or from the neuronal isoform of NOS (nNOS), which is located in some perivascular nerves (Yoshida, Okamura, Kimura, Bredt, Snyder & Toda, 1993; Davisson, Johnson & Lewis, 1994) as well as in fast-twitch skeletal muscle fibres (Nakane, Schmidt, Pollock, Förstermann & Murad, 1993; Kobzik, Reid, Bredt & Stamler, 1994; Brenman, Chao, Xia, Aldape & Bredt, 1995; Chang et al. 1996). Previous studies have demonstrated that the vascular endothelium releases NO in response to increased shear stress (Rubanyi, Romero & Vanhoutte, 1986), hypoxia (Pohl & Busse, 1989), or activation of endothelial α2-adrenergic receptors (Vanhoutte & Miller, 1989), all of which may occur in contracting skeletal muscle. Alternatively, a recent study in rats suggests that electrical stimulation of postganglionic lumbar sympathetic nerve terminals may release NO or nitrosyl factors such as S-nitrosothiols, resulting in hindlimb vasodilatation (Davisson et al. 1994). We believe that this mechanism is unlikely to explain our findings in the present study, since NOS inhibition potentiated sympathetic vasoconstriction in contracting hindlimb whether the vasoconstrictor stimulus was lumbar nerve stimulation or direct activation of α-adrenergic receptors. Finally, recent studies have shown that nNOS is highly abundant in rodent and human skeletal muscle. Because nNOS is a calcium-dependent enzyme, its activity is proportional to intracellular calcium which increases linearly with the intensity of skeletal muscle contraction. Within skeletal muscle, nNOS is preferentially localized to the sarcolemma of fast-twitch muscle fibres (Kobzik et al. 1994; Brenman et al. 1995; Chang et al. 1996), the same fibres in which we preferentially demonstrate functional sympatholysis (Thomas et al. 1994). In contrast, there is relatively little expression of nNOS in slow-twitch fibres (Kobzik et al. 1994) in which we can demonstrate little or no sympatholysis (Thomas et al. 1994).

The precise mechanism by which NO opposes adrenergic vasoconstriction is not known; however, our experiments suggest an interaction between NO and vascular KATP channels. We have shown previously in rats that KATP channel blockade alone partially reverses functional sympatholysis (Thomas et al. 1997). In the present study, NOS inhibition alone also partially reversed functional sympatholysis to a similar extent, but this effect was not further enhanced by KATP channel blockade. Taken together, one interpretation of these data is that functional sympatholysis may be mediated by NO-induced activation of KATP channels. As recently proposed by Murphy & Brayden (1995), NO-mediated increases in cGMP may activate a cGMP-dependent protein kinase that phosphorylates and activates the KATP channel. The resultant increase in potassium efflux would hyperpolarize the vascular smooth muscle, reducing calcium influx through voltage-sensitive calcium channels, thereby reducing sensitivity to vasoconstrictor stimuli. However, our data do not provide direct evidence for an interaction between NO and KATP channels. We therefore cannot exclude alternative explanations of the mechanism of action of NO such as a cGMP-dependent attenuation of receptor-mediated calcium influx in vascular smooth muscle, reduced calcium sensitivity of the contractile apparatus, enhanced cytosolic removal of calcium by Ca2+-ATPase, or activation of calcium-dependent potassium channels (Pohl & de Wit, 1996).

Despite its effect on functional sympatholysis, L-NAME in our experiments had no effect on contraction-induced hyperaemia per se. Although the effect of pharmacological inhibition of NOS on active hyperaemia has been studied both in experimental animals and in humans, the role of NO remains obscure (Green et al. 1996). Our findings in the present study suggest that by virtue of its ability to oppose sympathetic vasoconstriction, NO might assume a greater role in mediating active hyperaemia during moderate to heavy intensities of exercise, which in humans are accompanied by reflex increases in sympathetic nerve discharge (Mark, Victor, Nerhed & Wallin, 1985; Victor & Seals, 1989). Although this issue has not been studied directly, it recently has been shown in humans performing maximal cycle exercise that a close correlation exists between systemic noradrenaline levels and the venous concentration of nitrate and nitrite, which are the end products of NO metabolism (Node et al. 1997).

In conclusion, these results demonstrate that NO-mediated attenuation of sympathetic vasoconstriction is one important mechanism underlying functional sympatholysis in rat skeletal muscle. This effect of NO may be particularly important for the modulation of vasoconstriction mediated by α2-adrenergic receptors, which control the most distal, or nutrient, arterioles in rat skeletal muscle (Faber, 1988). Although the specific mechanism by which NO and sympathetic vasoconstriction interact is not known, it may involve NO-induced activation of vascular KATP channels. We speculate that these findings may have potential implications for understanding the impaired exercise capacity in pathophysiological conditions characterized by abnormalities of the NO system, such as peripheral atherosclerosis, diabetic vasculopathy, and heart failure.

Acknowledgments

We thank Dr Jere H. Mitchell for his continued support and critical review of our work. This research was supported by grants from the American Heart Association, Texas Affiliate (96G-064) and from the National Institutes of Health (P01-HL-06296).

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