Role of neuronal nitric oxide in the inhibition of sympathetic ...

J Appl Physiol 115: 97–106, 2013. First published May 2, 2013; doi:10.1152/japplphysiol.00250.2013.

Role of neuronal nitric oxide in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats Nicholas G. Jendzjowsky1 and Darren S. DeLorey1,2 1

Faculty of Physical Education and Recreation, University of Alberta, Edmonton, Alberta, Canada; and 2Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, Alberta, Canada Submitted 25 February 2013; accepted in final form 29 April 2013

Jendzjowsky NG, DeLorey DS. Role of neuronal nitric oxide in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats. J Appl Physiol 115: 97–106, 2013. First published May 2, 2013; doi:10.1152/japplphysiol.00250.2013.—Isoform-specific nitric oxide (NO) synthase (NOS) contributions to NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle are incompletely understood. The purpose of the present study was to investigate the role of neuronal NOS (nNOS) in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats. We hypothesized that acute pharmacological inhibition of nNOS would augment sympathetic vasoconstriction in resting and contracting skeletal muscle, demonstrating that nNOS is primarily responsible for NO-mediated inhibition of sympathetic vasoconstriction. Sprague-Dawley rats (n ⫽ 13) were anesthetized and instrumented with an indwelling brachial artery catheter, femoral artery flow probe, and lumbar sympathetic chain stimulating electrodes. Triceps surae muscles were stimulated to contract rhythmically at 60% of maximal contractile force. In series 1 (n ⫽ 9), the percent change in femoral vascular conductance (%FVC) in response to sympathetic stimulations delivered at 2 and 5 Hz was determined at rest and during muscle contraction before and after selective nNOS blockade with S-methyl-L-thiocitrulline (SMTC, 0.6 mg/kg iv) and subsequent nonselective NOS blockade with N␻-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg iv). In series 2 (n ⫽ 4), L-NAME was injected first, and then SMTC was injected to determine if the effect of L-NAME on constrictor responses was influenced by selective nNOS inhibition. Sympathetic stimulation decreased FVC at rest (⫺25 ⫾ 7 and ⫺44 ⫾ 8%FVC at 2 and 5 Hz, respectively) and during contraction (⫺7 ⫾ 3 and ⫺19 ⫾ 5%FVC at 2 and 5 Hz, respectively). The decrease in FVC in response to sympathetic stimulation was greater in the presence of SMTC at rest (⫺32 ⫾ 6 and ⫺49 ⫾ 8%FVC at 2 and 5 Hz, respectively) and during contraction (⫺21 ⫾ 4 and ⫺28 ⫾ 4%FVC at 2 and 5 Hz, respectively). L-NAME further increased (P ⬍ 0.05) the sympathetic vasoconstrictor response at rest (⫺47 ⫾ 4 and ⫺60 ⫾ 6%FVC at 2 and 5 Hz, respectively) and during muscle contraction (⫺33 ⫾ 3 and ⫺40 ⫾ 6%FVC at 2 and 5 Hz, respectively). The effect of L-NAME was not altered by the order of nNOS inhibition. These data demonstrate that NO derived from nNOS and endothelial NOS contribute to the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. sympathetic nervous system; nitric oxide synthase; exercise; blood flow THE PRESENCE OF TONIC SYMPATHETIC vasoconstriction in resting and contracting skeletal muscle has been well documented (5, 38). However, it is also well established that a number of substances released from skeletal muscle and/or vascular endothelium are able to inhibit sympathetic vasoconstriction in

Address for reprint requests and other correspondence: D. S. DeLorey, Faculty of Physical Education and Recreation, Univ. of Alberta, E-435 Van Vliet Centre, Edmonton, AB, Canada T6G 2H9 (e-mail: darren. [email protected]). http://www.jappl.org

resting and contracting skeletal muscle (13, 23, 39, 53). While the mechanism(s) responsible for the inhibition of sympathetic vasoconstriction has not been definitively established, the vasoactive molecule nitric oxide (NO) has been implicated (23, 31, 39, 53). NO is produced by the reaction of L-arginine with O2. The reaction is catalyzed by the enzyme NO synthase (NOS), which exists in three isoforms (48). The endothelial (eNOS) and neuronal (nNOS) isoforms of NOS are constitutively expressed in several tissues, including skeletal muscle and the endothelium (1, 4, 19, 32), and both isoforms appear to be involved in the regulation of skeletal muscle blood flow at rest and during exercise (9, 11, 48). The inducible NOS (iNOS) isoform is expressed in vascular tissues (60) but appears to be activated only during the immune response (44). Selective inhibition of iNOS did not alter skeletal muscle vascular conductance in healthy rabbits and humans, suggesting that iNOS was not involved in the control of vascular tone (18, 30). Nonselective pharmacological blockade of NOS has been shown to augment sympathetic vasoconstriction at rest and during exercise (23, 31, 39, 53). Unfortunately, nonselective NOS inhibition does not allow investigation of NOS isoformspecific blunting of sympathetic vasoconstriction. Removal or disruption of the endothelium (3, 14) has been shown to augment the vasoconstrictor response to norepinephrine in isolated blood vessels, and these findings have been interpreted as evidence for eNOS-mediated inhibition of sympathetic vasoconstriction at rest. However, nNOS is also expressed in the endothelium (1); therefore, removal of the endothelium may not exclusively affect eNOS-mediated NO production. NO derived from nNOS has been shown to contribute to the regulation of resting muscle blood flow (9, 46, 50, 52), and increased nNOS-mediated production of NO during muscle contraction has been documented (48). Recent studies that have selectively inhibited nNOS in rats indicate that nNOS contributes to the regulation of blood flow to oxidative skeletal muscles at rest (9) but not to exercise hyperemia during moderate-intensity exercise (9), but it does appear to be involved in the control of blood flow to glycolytic skeletal muscle during heavy-intensity exercise (11). A reduced ability to inhibit sympathetic vasoconstriction during muscle contraction has been reported in humans with Duchenne muscular dystrophy (43) and in genetically modified mice (50, 52) that do not express nNOS, suggesting that NO derived from nNOS may inhibit sympathetic vasoconstriction in contracting skeletal muscle. However, eNOS protein expression may also be reduced in genetically modified mice (35, 36), and whether findings from genetically modified animals and pathophysiological experimental models are reflective of vascular regulation in healthy individuals/animals has been questioned (28). Thus whether NO derived from eNOS, nNOS, or both is

8750-7587/13 Copyright © 2013 the American Physiological Society

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nNOS-Mediated Inhibition of Sympathetic Vasoconstriction

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Jendzjowsky NG et al.

responsible for the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle has not been clearly established, and further investigation is warranted. Acute isoform-specific pharmacological inhibition of NOS allows investigation of isoform-specific effects on sympathetic vasoconstriction in healthy animals without acute or chronic alterations to vascular structures and other signaling pathways. Therefore, the purpose of the present study was 1) to investigate nNOS-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle via selective nNOS blockade with the nNOS-specific antagonist S-methylL-thiocitrulline (SMTC) and 2) to establish the relative contribution of nNOS to the total amount of NO-mediated inhibition of sympathetic vasoconstriction in healthy wild-type rats. It was hypothesized that NO derived from nNOS would inhibit sympathetic vasoconstriction in resting and contracting muscle and that nNOS would be the primary mediator of NO-dependent inhibition of sympathetic vasoconstriction.

calcaneal tendon. Maximal contractile force (MCF) was determined by stimulation of the triceps surae muscle group with twenty-five 1-ms impulses delivered at 100 Hz and 10⫻ motor threshold. For determination of the optimal muscle length for tension development, the muscle was progressively lengthened and the nerve stimulation was repeated until a plateau in tension (peak ⫺ baseline) was observed. Rhythmic contractions of the triceps surae muscle group at 60% MCF were produced by stimulation of the sciatic nerve with trains of electrical stimulation (40 Hz, 0.1-ms pulses, in 250-ms trains at a rate of 60 trains/min at ⬃5⫻ motor threshold) delivered through integrated stimulation software (Chart 7.2, ADInstruments). We previously documented NO-mediated inhibition of sympathetic vasoconstriction during muscle contraction at 30 and 60% MCF (31). However, the magnitude of NO-mediated inhibition of vasoconstriction was contraction intensity-dependent (31). Thus we reasoned that our ability to detect differences in nNOS- and eNOS-mediated inhibition of sympathetic vasoconstriction in contracting muscle would be greatest at a contractile intensity of 60% MCF.

METHODS

Through a laparotomy, the lumbar chain distal to the renal branch was dissected free with a blunt glass pipette. A bipolar silver-wire stimulating electrode was attached to the lumbar sympathetic chain between L3 and L4. The electrodes were secured in place and electrically isolated by embedding them in a rapidly curing nontoxic silicone elastomer (Kwiksil, WPI, Sarasota, FL). An isolated constantcurrent stimulator (model DS3, Digitimer, Welwyn City, UK) was used to deliver 1 min of 1-ms, 1-mA pulses of stimulation at 2 and 5 Hz in random order.

Animals and Animal Care Male Sprague-Dawley rats (n ⫽ 13, 10 –12 wk old) were obtained from the institutional breeding colony. They were housed in pairs in a 12:12-h light-dark cycle, environmentally controlled (22–24°C, 40 –70% humidity) room. Water and rat chow (Lab Diet 5001, PMI Nutrition, Brentwood, MO) were provided ad libitum. All experiments were conducted in accordance with the Canadian Council on Animal Care Guidelines and Policies with approval from the Animal Care and Use Committee: Health Sciences for the University of Alberta. Instrumentation Anesthesia was induced by inhalation of 3–3.5% isoflurane-balance O2. The right jugular vein was then cannulated, and anesthesia was maintained with infusion of ␣-chloralose (8 –16 mg·kg⫺1·h⫺1) and urethane (50 –100 mg·kg⫺1·h⫺1). The depth of anesthesia was assessed by the stability of arterial blood pressure and heart rate (HR) and the absence of a withdrawal reflex in response to a painful stimulus (i.e., paw pinch). A tracheotomy was performed to allow spontaneous breathing of room air. We previously demonstrated that arterial blood gases and acid-base status are well maintained at rest and during muscle contraction in this preparation (31). Thus arterial blood gases were checked periodically to confirm maintenance of blood gas and acid-base status in this study (IDEXXLaboratories, VetStat, Markham, ON, Canada). Arterial blood pressure was measured by a solid-state pressure transducer (Abbott, North Chicago, IL) that was attached to a cannula implanted in the left brachial artery. HR was derived from the blood pressure waveform. The left femoral artery and vein were cannulated for delivery of pharmacology. Blood flow was measured continuously using a transit-time flow probe (model 0.7V, Transonic Systems, Ithaca, NY) placed around the right femoral artery and connected to a flowmeter (model T106, Transonic Systems). Core temperature was monitored by a rectal probe and maintained at 36 –37°C by an external heating pad (model TCAT-2, Physitemp, Clifton, NJ). On completion of all experiments, animals were euthanized by anesthetic overdose with ␣-chloralose and urethane. Muscle Contraction The right sciatic nerve was exposed and instrumented with a cuff electrode. The triceps surae muscle group was then dissected free of all skin and connective tissue and attached to a force transducer (model MLT1030/D, ADInstruments, Colorado Springs, CO) via the

Lumbar Sympathetic Chain Stimulation

Experimental Procedures Series 1. After the surgical procedure, a 20-min recovery period was used to establish baseline hemodynamic values. The cardiovascular response [change in mean arterial pressure (MAP), HR, femoral arterial blood flow (FBF), and femoral vascular conductance (FVC)] to sympathetic stimulation was then determined at rest and during contraction at 60% MCF (n ⫽ 9, 353 ⫾ 27 g). Sympathetic stimulations were delivered in a random order in resting skeletal muscle with sufficient time (⬃2 min) allowed between stimulations to restore baseline hemodynamic values. Bouts of muscle contraction were 8 min in duration. During muscle contraction, the lumbar sympathetic chain was stimulated at 2 and 5 Hz in random order 3 and 6 min after the onset of contraction. Rats were allowed to recover for ⬃20 min, and then the selective nNOS antagonist SMTC (0.6 mg/kg iv) was injected. After stabilization of hemodynamic variables (⬃20 min), lumbar sympathetic chain stimulations were repeated at rest and during skeletal muscle contraction. After another period of recovery (⬃20 min), the nonselective NOS antagonist N␻-nitro-L-arginine methyl ester hydrochloride (LNAME, 5 mg/kg iv) was injected. After hemodynamic stabilization (⬃20 min), lumbar sympathetic chain stimulations were repeated at rest and during skeletal muscle contraction. We previously demonstrated that muscle force production and the cardiovascular response to sympathetic stimulation are not altered over time when bouts of contraction are repeated in this manner (31). Effectiveness of NOS blockade. To assess the effectiveness of selective nNOS and nonselective NOS blockade, intra-arterial bolus injections of ACh (0.005 and 0.1 ␮g) were administered prior to and following SMTC and L-NAME. Injections were delivered in small volumes (100 ␮l) over ⬃5 s to avoid any flow-mediated vasodilation and separated by ⬃5 min. Vehicle injections delivered in this manner did not alter FBF. The dose of SMTC was selected on the basis of previous investigations that showed effective blockade of nNOS at similar doses of SMTC (29, 45, 46, 56) and preliminary experiments in our laboratory. In preliminary experiments, we determined the highest dose of SMTC

J Appl Physiol • doi:10.1152/japplphysiol.00250.2013 • www.jappl.org


Drug Condition

HR, beats/min

MAP, mmHg

FBF, ml/min

FVC, ml 䡠 min⫺1 䡠 mmHg⫺1

Control SMTC L-NAME

383 ⫾ 39 382 ⫾ 38 329 ⫾ 23*‡

84 ⫾ 8† 99 ⫾ 8† 132 ⫾ 11†

3.4 ⫾ 0.6 3.6 ⫾ 0.4 3.6 ⫾ 0.7

0.041 ⫾ 0.009† 0.037 ⫾ 0.005† 0.028 ⫾ 0.006†

Values are means ⫾ SD. HR, heart rate; MAP, mean arterial blood pressure; FBF, femoral blood flow; FVC, femoral vascular conductance; SMTC, Smethyl-l-thiocitrulline; l-NAME, N␻-nitro-l-arginine methyl ester. *P ⬍ 0.05 vs. baseline. †P ⬍ 0.05 between all drug conditions. ‡P ⬍ 0.05 vs. SMTC.

that would induce a pressor response and a decrease in vascular conductance but no effect on ACh-mediated vasodilation. SMTC was administered at 0.2–1.5 mg/kg, and systemic hemodynamics and the response to a bolus infusion of ACh and lumbar sympathetic stimulation were assessed. At 0.4 – 0.6 mg/kg, SMTC induced a pressor response, decreased vascular conductance, and augmented the constrictor response to sympathetic stimulation without affecting vasodilation to ACh. At ⬎0.6 mg/kg, SMTC inhibited vasodilation to ACh. Thus, on the basis of the preliminary data, SMTC at 0.6 mg/kg appears to selectively inhibit nNOS without inhibiting eNOS, consistent with other recent studies (9 –11, 29, 45, 46, 56). Series 2. In an additional group of animals (n ⫽ 4, 371 ⫾ 46 g), the order of NOS antagonist administration was reversed, with L-NAME injected first followed by SMTC. These experiments were completed to determine whether the magnitude of NO-mediated inhibition of sympathetic vasoconstriction in the L-NAME condition was affected by prior SMTC administration and to provide additional evidence of selective nNOS blockade. After surgical instrumentation and recovery, sympathetic stimulations were delivered in random order at 2 and 5 Hz at rest and during muscle contraction at 60% MCF. After ⬃20 min of recovery, L-NAME (5 mg/kg iv) was administered, hemodynamic parameters were allowed to stabilize, and lumbar sympathetic chain stimulations were repeated at rest and during skeletal muscle contraction. After another period of recovery, the nNOS antagonist SMTC (0.6 mg/kg iv) was injected, and lumbar sympathetic chain stimulations were repeated at rest and during skeletal muscle contraction. Data Analysis Data were recorded using Chart 7.2 data acquisition software (ADInstruments). Arterial blood pressure and FBF were sampled at 100 Hz, and FVC was calculated. Peak force and fatigue index {[(peak force ⫺ end-contraction force) ⫼ peak force] ⫻ 100} were calculated for each contractile bout. The change in HR, MAP, FBF, and FVC in response to sympathetic stimulation was calculated as an absolute change and as a percent change from the value preceding the sympathetic stimulation. The percent change in FVC is the accepted metric to assess the magnitude of sympathetic vasoconstrictor responses, because the percent change in FVC accurately reflects percent changes in resistance vessel radius even across conditions with different baseline levels of vascular conductance (5, 51). Thus the magnitude of sympathetic vasoconstrictor responses to lumbar chain stimulation was assessed by percent changes in FVC. The effect of SMTC and L-NAME on the magnitude of sympathetic vasoconstriction (decrease in %FVC) was determined by calculating the difference between sympathetic vasoconstriction achieved during control, SMTC, and L-NAME conditions. The response to ACh was calculated as the difference between the peak FVC response (⬃3 s average) and the preinfusion baseline (⬃20 s average) and expressed as percent change from the FVC baseline. Values are means ⫾ SD. Statistics The vasoconstrictor response to sympathetic stimulation during all conditions was analyzed by two-way repeated-measures ANOVA

99


(muscle contractile state ⫻ drug condition). Differences in baseline hemodynamics, the hemodynamic response to contraction, contractile force, and the vasodilator responses to ACh were analyzed by oneway repeated-measures ANOVA. The effect of muscle contractile state on the contribution of nNOS to the total NO-mediated inhibition of sympathetic vasoconstriction was determined with a paired t-test. When significant F-ratios were detected, Student-Newman-Keuls post hoc testing was completed. P ⬍ 0.05 was considered statistically significant. RESULTS

Resting Hemodynamics Resting hemodynamics are presented in Table 1. Resting HR was unchanged by SMTC but was decreased (P ⬍ 0.05) by the subsequent addition of L-NAME (Table 1). MAP was increased (P ⬍ 0.05) by SMTC and increased (P ⬍ 0.05) further by L-NAME (Table 1). FBF was not different (P ⬎ 0.05) between conditions; however, FVC was reduced (P ⬍ 0.05) by SMTC and further reduced (P ⬍ 0.05) by addition of L-NAME (Table 1). Effectiveness of NOS Blockade ACh-mediated vasodilation was unaffected by SMTC (P ⬎ 0.05), consistent with the dosing regimen outlined in METHODS (Fig. 1). In contrast, the response to ACh was reduced (P ⬍ 0.05) by L-NAME (Fig. 1). Series 1 Sympathetic vasoconstriction at rest and during muscle contraction. The response to sympathetic stimulation delivered at rest and during muscle contraction in a representative rat is shown in Fig. 2. The absolute changes in HR, MAP, FBF, and FVC in response to sympathetic stimulation at rest and during contraction are presented in Table 2. A main effect (P ⬍ 0.05) of drug treatment and muscle contractile state on the magnitude of vasoconstriction (%decrease in FBF and FVC) in response to sympathetic stimulation delivered at 2 and 5 Hz was observed. There was also a significant interaction (P ⬍ 0.05) between drug condition and muscle contractile state. In resting skeletal muscle, post hoc testing demonstrated that the sympa-

100

0.005µg ACh 0.1µg ACh

FVC (% Change)

Table 1. Resting hemodynamics

•

80 60

^

40 20 0

Control

SMTC

SMTC + L-NAME

Fig. 1. Percent changes in femoral vascular conductance (FVC) in response to intra-arterial bolus injections of ACh (0.005 and 0.1 ␮g). L-NAME, N␻-nitroL-arginine methyl ester; SMTC, S-methyl-L-thiocitrulline. Values are means ⫾ SD. ^P ⬍ 0.05 vs. control. This symbol indicates a difference from control and SMTC conditions. Please have it read: ^P ⬍ 0.05 vs. control and SMTC.


100

•


A

B

Contractile force (g)

Fig. 2. Original data from a representative animal illustrating the response of mean arterial blood pressure (MAP), femoral artery blood flow (FBF), FVC, and muscle contractile force at rest (A) and during muscle contraction (B). Arrow indicates onset of contraction. Lumbar sympathetic nerve stimulations were delivered at 2 and 5 Hz in random order at rest and during contraction.

FVC (mL·min-1·mmHg-1)

FBF (mL·min-1)

MAP (mmHg)


Time (s)

thetic vasoconstrictor response was different between all drug conditions, with the constrictor response being augmented (P ⬍ 0.05) by SMTC and further increased (P ⬍ 0.05) by L-NAME (Fig. 3).

Table 2. Hemodynamic responses to sympathetic stimulation at rest and during muscle contraction HR, beats/min

MAP, mmHg

FBF, ml/min


2 Hz Rest Control SMTC L-NAME Contraction Control SMTC L-NAME Rest Control SMTC L-NAME Contraction Control SMTC L-NAME

⫺15 ⫾ 7 ⫺9 ⫾ 6 ⫺7 ⫾ 5

6⫾5 4⫾3 9 ⫾ 2*

⫺0.7 ⫾ 0.3‡ ⫺0.0105 ⫾ 0.005 ⫺1.1 ⫾ 0.2‡ ⫺0.0120 ⫾ 0.004 ⫺1.6 ⫾ 0.4‡ ⫺0.0132 ⫾ 0.004

⫺10 ⫾ 7 ⫺13 ⫾ 13 ⫺7 ⫾ 8

2⫾3 4⫾3 10 ⫾ 6*

⫺0.3 ⫾ 0.3‡ ⫺0.0051 ⫾ 0.003‡ ⫺1.5 ⫾ 0.4‡ ⫺0.0188 ⫾ 0.005‡ ⫺2.7 ⫾ 0.8‡ ⫺0.0253 ⫾ 0.007‡ 5 Hz

⫺22 ⫾ 14 ⫺21 ⫾ 8 ⫺16 ⫾ 10

15 ⫾ 7 12 ⫾ 7 13 ⫾ 4

⫺1.2 ⫾ 0.5‡ ⫺0.0184 ⫾ 0.007 ⫺1.7 ⫾ 0.5‡ ⫺0.0200 ⫾ 0.006 ⫺2.2 ⫾ 0.6‡ ⫺0.0181 ⫾ 0.005

⫺24 ⫾ 10 ⫺22 ⫾ 9 ⫺12 ⫾ 17

11 ⫾ 6 7⫾5 19 ⫾ 6*

⫺0.6 ⫾ 0.6‡ ⫺0.0150 ⫾ 0.005‡ ⫺2.0 ⫾ 0.6‡ ⫺0.0253 ⫾ 0.005‡ ⫺3.0 ⫾ 0.8‡ ⫺0.0309 ⫾ 0.008‡

Values are means ⫾ SD. *P ⬍ 0.05 vs. control and SMTC. ‡P ⬍ 0.05 between all drug conditions within the specified contractile state.

Compared with rest, muscle contraction attenuated the vasoconstrictor response to sympathetic stimulation at 2 and 5 Hz (P ⬍ 0.05) in all drug conditions (Fig. 3). During muscle contraction, the sympathetic vasoconstrictor response (%decrease in FBF and FVC) was different (P ⬍ 0.05) between all drug conditions, with the constrictor response being increased (P ⬍ 0.05) above the response under control conditions by selective nNOS inhibition (SMTC) and further increased (P ⬍ 0.05) by nonselective NOS inhibition (L-NAME; Fig. 3). However, the magnitude of contraction-induced blunting of sympathetic vasoconstrictor responsiveness was dependent on the drug condition (interaction, P ⬍ 0.05). The contraction-induced reduction in the sympathetic vasoconstrictor response was larger under control conditions than with SMTC and SMTC ⫹ L-NAME treatment at 2 Hz (difference between means: 19%, 11%, and 15% for control, SMTC, and SMTC ⫹ L-NAME, respectively) and 5 Hz (difference between means: 25%, 22%, and 21% for control, SMTC, and SMTC ⫹ LNAME, respectively), indicating less blunting of sympathetic vasoconstriction by muscle contraction during selective and nonselective NOS blockade. In resting skeletal muscle, nNOS was responsible for ⬃30% (31 ⫾ 17% and 33 ⫾ 12% at 2 and 5 Hz, respectively) of the total NO-mediated inhibition of sympathetic vasoconstriction. In contracting skeletal muscle, nNOS-mediated inhibition of sympathetic vasoconstriction increased (P ⬍ 0.05) and was responsible for ⬃50% of total NO-mediated inhibition of



FBF (% Change)

Rest


101

60% MCF

0

0

-20

-20

-40

-40

-60

‡

Fig. 3. Percent change in FBF and FVC in response to sympathetic stimulation at 2 and 5 Hz in resting and contracting muscle [60% maximal contractile force (MCF)] under control conditions, following selective neuronal nitric oxide synthase (nNOS) blockade with SMTC (0.6 mg/kg iv), and following nonselective NOS blockade with L-NAME (5 mg/kg iv). Values are means ⫾ SD. ‡P ⬍ 0.05 between all drug conditions in response to 2- and 5-Hz sympathetic stimulation at rest and during muscle contraction.

‡

-60 ‡

-80

FVC (% Change)

•

-80

‡

0

0

-20

-20

-40

-40

-60

-60

‡ ‡

2Hz 5Hz

‡ -80

-80 ‡ Control

SMTC

SMTC + L-NAME

Control

SMTC

sympathetic vasoconstriction (56 ⫾ 19% and 47 ⫾ 9% at 2 and 5 Hz, respectively; Fig. 4). Muscle force production and hyperemic response to contraction. Muscle force production (1,125 ⫾ 105, 1,038 ⫾ 99, and 1,014 ⫾ 65 g for control, SMTC, and L-NAME, respectively) and fatigue index (52 ⫾ 6%, 54 ⫾ 10%, and 53 ⫾ 6% for control, SMTC, and L-NAME, respectively) were not different (P ⬎ 0.05) between conditions. The increase in HR, MAP, FBF, and FVC in response to contraction were not different (P ⬎ 0.05) between conditions (Table 3). Series 2 In resting skeletal muscle, the magnitude of vasoconstriction in response to sympathetic stimulation delivered at 2 and 5 Hz was augmented (P ⬍ 0.05) by L-NAME. After the injection of SMTC, the response to sympathetic stimulation was unchanged from the L-NAME condition (Fig. 5; P ⬎ 0.05). During muscle contraction, L-NAME increased (P ⬍ 0.05) the magnitude of vasoconstriction in response to sympathetic stimulation. Consistent with the resting data, the magnitude of vasoconstriction in response to sympathetic stimulation was not different (P ⬎ 0.05) between the L-NAME and L-NAME ⫹ SMTC conditions (Fig. 5). DISCUSSION

The purpose of the present study was to investigate whether NO derived from nNOS inhibited sympathetic vasoconstriction in resting and contracting skeletal muscle. We also sought to determine the relative contribution of NO derived from nNOS to total NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. The present study demonstrated that acute pharmacological inhibition of nNOS augmented sympathetic vasoconstriction in resting and contracting skeletal muscle, indicating that nNOS

SMTC + L-NAME

contributes to NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. Several laboratories, including our own (31), have shown that NO inhibits sympathetic vasoconstriction at rest and during muscle contraction (3, 6, 14, 23, 53). However, there has been limited investigation of specific nNOS- and eNOS-mediated blunting of sympathetic vasoconstriction in healthy animals/humans, and the available literature is conflicting. Evidence to support nNOS-mediated inhibition of sympathetic vasoconstriction has been garnered from experimental models where nNOS expression was reduced or absent (16, 43, 50, 52). For example, a diminished ability to inhibit sympathetic vasoconstriction during handgrip exercise has been reported in humans with Duchenne muscular dystrophy (43). A loss of estrogen also appears to reduce nNOS expression (57, 58), and a reduced inhibition of sympathetic vasoconstriction has been reported in ovariectomized mice (17) and postmenopausal women (16). Previous studies in nNOS-null mice have also shown a reduced inhibition of sympathetic vasoconstriction during muscle contraction compared with wild-type C57BL/6 mice (50, 52). Infusion of L-NAME in nNOS-null mice did not increase sympathetic vasoconstriction during muscle contraction (50, 52), suggesting that NO-mediated inhibition of sympathetic vasoconstriction may be mediated entirely by NO derived from nNOS. In contrast, studies where the vascular endothelium was removed or disrupted have provided evidence of eNOS-mediated inhibition of sympathetic vasoconstriction (3, 14, 39). The underlying assumption in these studies was that removal of the endothelium would interrupt eNOS-mediated NO production and that subsequent nonselective NOS inhibition with L-NAME would block nNOS-mediated NO production. However, nNOS has also been shown to be expressed in the endothelium (1); thus it is possible that removal of the endo-


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A

80

nNOS Component (%)

Increase in Sympathetic Vasoconstriction (%FVC)

Rest

•

30

20

10

0

60

40

20

0

60% MCF

80

30

nNOS Component (%)

Increase in Sympathetic Vasoconstriction (%FVC)

Fig. 4. A: magnitude of increase in sympathetic vasoconstriction during selective nNOS inhibition and nonselective NOS inhibition at rest and during muscle contraction (60% MCF). B: contribution of NO derived from nNOS to total NO-mediated inhibition of sympathetic vasoconstriction at rest and during muscle contraction. Values are means ⫾ SD. †P ⬍ 0.05 vs. rest.

B

20

10

† †

60

40

20

0

0 2 Hz

5 Hz

2 Hz

Stimulation Frequency

5 Hz

Stimulation Frequency

nNOS Blockade

60% MCF

Rest

Non-Selective nNOS Blockade

60% MCF

60% MCF

thelium also affected nNOS-mediated NO production, and the contribution of nNOS-derived NO to the inhibition of sympathetic vasoconstriction may have been underestimated in these experiments. Nonetheless, after disruption of the endothelium, a larger constrictor response to sympathetic stimulation was observed in isolated arterioles from the cremaster muscle (39) vascular beds. Subsequent nonselective NOS inhibition did not further increase vasoconstriction, suggesting that the inhibition of sympathetic vasoconstriction was mediated entirely by eNOS (39). However, in the rat mesenteric vascular bed, the pressor response to perivascular nerve stimulation was increased following disruption of the endothelium, and subsequent nonselective NOS blockade further increased the pressor response to Table 3. Hemodynamic response to muscle contraction Drug Condition

HR, beats/min

MAP, mmHg

FBF, ml/min


Control SMTC L-NAME

7⫾5 9⫾3 10 ⫾ 4

4⫾7 2⫾6 0⫾7

4.7 ⫾ 0.7 4.5 ⫾ 0.9 4.6 ⫾ 0.1

0.043 ⫾ 0.005 0.045 ⫾ 0.008 0.041 ⫾ 0.008

Values (means ⫾ SD) represent the absolute increase of each variable from rest in response to muscle contraction.

nerve stimulation, suggesting that NO derived from eNOS and nNOS may inhibit sympathetic vasoconstriction (41). The present study utilized highly specific nNOS blockade with SMTC (21) to assess nNOS-mediated inhibition of sympathetic vasoconstriction. Sympathetic vasoconstrictor responsiveness was augmented at rest and during contraction following the infusion of SMTC, demonstrating that NO derived from nNOS inhibited sympathetic vasoconstriction in resting and contracting skeletal muscle. Subsequent infusion of L-NAME following selective nNOS blockade by SMTC resulted in a further increase in the vasoconstrictor response to sympathetic stimulation in resting and contracting skeletal muscle, demonstrating that NO derived from nNOS and eNOS contributes to NO-mediated inhibition of sympathetic vasoconstriction. Although iNOS is expressed in vascular tissue (60), selective inhibition of iNOS did not alter skeletal muscle vascular conductance (18, 30). Therefore, it is unlikely that iNOS contributed to the acute regulation of skeletal muscle blood flow or the inhibition of sympathetic vasoconstriction at rest or during contraction in the present study. Thus we believe that the increase in vasoconstriction in the L-NAME condition represents the total magnitude of NO-mediated inhibition of sympathetic vasoconstriction and that the difference between the constrictor response in the SMTC and L-NAME conditions



FBF (% Change)

Rest

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60% MCF

0

0

-20

-20

-40

-40

^ ^

-60

-60

^

Fig. 5. Percent change in FBF and FVC in response to sympathetic stimulation at 2 and 5 Hz in resting and contracting (60% MCF) muscle under control conditions, following nonselective NOS blockade with L-NAME (5 mg/kg iv), and following L-NAME ⫹ selective nNOS blockade with SMTC (0.6 mg/kg iv). Values are means ⫾ SD. ^P ⬍ 0.05 vs. control.

^

FVC (% Change)

103

-80

-80

0

0

-20

-20

-40

-40 ^

-60

^

-60

2Hz 5Hz

^ ^

-80 Control

L-NAME

-80 L-NAME + SMTC

Control

L-NAME

represents eNOS-mediated inhibition of sympathetic vasoconstriction. Therefore, the present data suggest that both nNOS and eNOS contribute to the inhibition of sympathetic vasoconstriction. In resting skeletal muscle, we estimate that nNOS was responsible for ⬃30% of NO-mediated inhibition of sympathetic vasoconstriction; whereas during contraction, the contribution of nNOS to NO-mediated inhibition of sympathetic vasoconstriction increased to ⬃50% (Fig. 4). A relatively small contribution of nNOS to the inhibition of sympathetic vasoconstriction in resting skeletal muscle is consistent with evidence indicating that the tonic production of NO at rest is predominantly attributable to eNOS (27). In agreement with this finding, Grange et al. (22) demonstrated that NO derived from eNOS was predominantly responsible for the regulation of vascular tone at rest in eNOS- and nNOS-null mice. An increased contribution of NO derived from nNOS to the inhibition of sympathetic vasoconstriction in contracting skeletal muscle suggests that contraction induced an upregulation of nNOS-mediated NO production. The increase in skeletal muscle intracellular Ca2⫹ concentration during contraction is thought to activate nNOS, leading to novel NO production (2). Consistent with this notion, nNOS-mediated production of NO appears to be required for the formation of cGMP and relaxation of vascular smooth muscle during muscle contraction (22, 33). Indeed, a similar release of NO during contraction has been shown in eNOS-null mice and C57BL/6 control mice, suggesting that eNOS does not contribute to NO production during muscle contraction (27). Moreover, an impaired ability to inhibit sympathetic vasoconstriction has been reported in nNOS-null or nNOS-deficient populations (17, 22, 43, 50), further indicating that novel NO derived from nNOS may be required to inhibit sympathetic vasoconstriction in contracting muscle, consistent with the findings from the present study.

L-NAME + SMTC

Involvement of nNOS in the Regulation of Blood Flow to Resting and Contracting Skeletal Muscle Consistent with previous investigations that have argued in favor of a role for nNOS in the tonic regulation of vascular conductance in resting skeletal muscle (9, 46, 50, 52), selective nNOS inhibition by SMTC augmented MAP and reduced FVC at rest in the present study. In agreement with previous investigations, subsequent nonspecific NOS inhibition with L-NAME further increased arterial blood pressure and reduced FVC in resting skeletal muscle in the present study, indicating that NO derived from eNOS contributes to the regulation of vascular tone in resting skeletal muscle (47, 55). The increase in HR, MAP, FBF, and FVC in response to muscle contraction at 60% MCF was not different between control and SMTC conditions in the present study. In previous investigations utilizing acute selective pharmacological blockade of nNOS, nNOS was not required for contraction-induced hyperemia during moderate-intensity exercise (9), whereas nNOS inhibition reduced blood flow to glycolytic muscles during heavy-intensity treadmill exercise (11). L-NAME did not alter the increase in HR, MAP, FBF, and FVC in response to contraction in the present study, suggesting that neither nNOS- nor eNOS-derived NO was required for exercise hyperemia. Several previous studies have reported that NO does not contribute to exercise hyperemia (15, 20, 42, 59), whereas others have shown that NOS inhibition reduced skeletal muscle FBF or FVC during exercise (12, 26, 37). Collectively, the present findings that NO inhibited sympathetic vasoconstriction in contracting skeletal muscle but was not required for the hyperemic response to contraction may appear paradoxical. However, other investigators have suggested that the functional role of NO-mediated inhibition of sympathetic vasoconstriction in the overall regulation of tissue blood flow is to optimize the distribution of blood flow be-


104


tween and within skeletal muscles, while also facilitating the maintenance of systemic arterial blood pressure (51, 54). The present experimental approach where the entire hindlimb was stimulated to contract precludes determination of muscle/fiber type-specific inhibition of sympathetic vasoconstriction and the distribution of muscle blood flow. However, different degrees of NO-mediated inhibition of sympathetic vasoconstriction may have occurred in different muscles/fiber types, and prior studies have reported that the inhibition of sympathetic vasoconstriction may occur predominantly in glycolytic muscle (49). Experimental Considerations and Limitations A major strength of the present investigation was the use of acute selective nNOS inhibition to investigate NO-mediated inhibition of sympathetic vasoconstriction in healthy animals. Previous investigations of isoform-specific NOS vascular regulation have largely relied on genetically modified nNOS- and eNOS-null mice or pathophysiological human models of Duchenne muscular dystrophy with reduced NOS expression (33, 50, 52). While these studies provide valuable data, it is possible that these experimental models also have altered skeletal muscle development and/or that other vascular signaling pathways or structures are also affected by the genetic deletion or chronic reduction of NO bioavailability (28, 34, 35). We utilized SMTC, a nNOS-specific antagonist (21), and the subsequent infusion of L-NAME, a nonspecific NOS antagonist, to investigate the roles of nNOS and eNOS in NO-mediated inhibition of sympathetic vasoconstriction. The interpretation of the present experimental data is based on the premise that nNOS was completely inhibited by SMTC and that subsequent injection of L-NAME completely blocked all NOS. If selective or total NOS inhibition was incomplete, the relative contributions of nNOS and eNOS to the inhibition of sympathetic vasoconstriction and the total magnitude of NO-mediated inhibition may be underestimated. We believe that this is unlikely or represents a very small error. SMTC has an affinity for nNOS ⬃17 times that of eNOS (21) and does not appear to affect efferent sympathetic nerve discharge (10), endothelial function, or eNOS (21) at the dose utilized in the present study. Consistent with other investigations (8, 9, 11), the vasodilator response to ACh was similar between the control and SMTC conditions in the present study, suggesting that SMTC selectively blocked nNOS without inhibiting eNOS. In contrast, nonselective NOS blockade with L-NAME significantly reduced ACh-mediated vasodilation, in agreement with previous reports (40). The selective inhibition of nNOS by SMTC is further evidenced by the increased vasoconstrictor response when L-NAME treatment was added to selective nNOS inhibition with SMTC. The observation of a similar vasoconstrictor response to sympathetic stimulation in the L-NAME condition (series 2) and the SMTC ⫹ L-NAME (series 1) and L-NAME ⫹ SMTC (series 2) conditions suggests that NOS was completely blocked by L-NAME in the present study. The dose of LNAME utilized in the present study also produced a pressor response and decrease in vascular conductance that were similar in magnitude to those observed in previous studies that employed much higher doses of L-NAME, further indicating complete NOS inhibition at the L-NAME dose utilized in the present study (23, 25, 26, 37).

•


Finally, whether NO inhibits sympathetic vasoconstriction in humans remains controversial, because the available evidence is conflicting (6, 13, 43, 54, 59). Thus further experimentation is required to determine whether the present findings in rats are reflective of NO-mediated vascular control of skeletal muscle blood flow in humans. Conclusion The current data demonstrate that NO produced by nNOS inhibited sympathetic vasoconstriction in resting and contracting skeletal muscle. It appears that eNOS and nNOS contribute to NO production at rest and during contraction and are necessary for the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. However, it appears that, during muscle contraction, newly derived NO from nNOS is necessary to increase the inhibition of sympathetic vasoconstriction in the face of elevated sympathetic outflow. Altered nNOS regulation of skeletal muscle blood flow has been reported in aging and heart failure (7, 24). Thus therapies and treatments designed to augment the expression and/or localization of nNOS may improve the inhibition of sympathetic vasoconstriction and the control of skeletal muscle blood flow at rest and during exercise in health and disease. GRANTS This project was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Foundation for Innovation, and Alberta Advanced Education and Technology. N. G. Jendzjowsky was supported by a NSERC Graduate Doctoral Scholarship, University of Alberta Presidents’ Scholarship, and Izaak Walton Killam Memorial Scholarship. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS N.G.J. and D.S.D. are responsible for conception and design of the research; N.G.J. performed the experiments; N.G.J. and D.S.D. analyzed the data; N.G.J. and D.S.D. interpreted the results of the experiments; N.G.J. and D.S.D. prepared the figures; N.G.J. and D.S.D. drafted the manuscript; N.G.J. and D.S.D. edited and revised the manuscript; N.G.J. and D.S.D. approved the final version of the manuscript. REFERENCES 1. Bachetti T, Comini L, Curello S, Bastianon D, Palmieri M, Bresciani G, Callea F, Ferrari R. Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells. J Mol Cell Cardiol 37: 939 –945, 2004. 2. Balon TW, Nadler JL. Nitric oxide release is present from incubated skeletal muscle preparations. J Appl Physiol 77: 2519 –2521, 1994. 3. Behnke BJ, Armstrong RB, Delp MD. Adrenergic control of vascular resistance varies in muscles composed of different fiber types: influence of the vascular endothelium. Am J Physiol Regul Integr Comp Physiol 301: R783–R790, 2011. 4. Boulanger CM, Heymes C, Benessiano J, Geske RS, Levy BI, Vanhoutte PM. Neuronal nitric oxide synthase is expressed in rat vascular smooth muscle cells: activation by angiotensin II in hypertension. Circ Res 83: 1271–1278, 1998. 5. Buckwalter JB, Clifford PS. The paradox of sympathetic vasoconstriction in exercising skeletal muscle. Exerc Sport Sci Rev 29: 159 –163, 2001. 6. Chavoshan B, Sander M, Sybert TE, Hansen J, Victor RG, Thomas GD. Nitric oxide-dependent modulation of sympathetic neural control of oxygenation in exercising human skeletal muscle. J Physiol 540: 377–386, 2002. 7. Copp SW, Hirai DM, Ferguson SK, Holdsworth CT, Musch TI, Poole DC. Effects of chronic heart failure on neuronal nitric oxide synthase-



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