Sage Journals: Discover world-class research

Abstract

The nitric oxide synthase (NOS)-independent pathway of nitric oxide (NO) production in which nitrite (NO₂⁻) is reduced to NO may have therapeutic applications for those with cardiovascular diseases in which the NOS pathway is downregulated. We tested the hypothesis that NO₂⁻ infusion would reduce mean arterial pressure (MAP) and increase skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise in the face of NOS blockade via L-NAME. Following infusion of L-NAME (10 mg kg⁻¹, L-NAME), male Sprague-Dawley rats (3-6 months, n = 8) exercised without N^G-nitro-L arginine methyl ester (L-NAME) and after infusion of sodium NO₂⁻ (7 mg kg⁻¹; L-NAME + NO₂⁻). MAP and hindlimb skeletal muscle BF (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m min⁻¹, 5% grade). Across group comparisons were made with a published control data set (n = 11). Relative to L-NAME, NO₂⁻ infusion significantly reduced MAP (P < 0.03). The lower MAP in L-NAME+NO₂⁻ was not different from healthy control animals (control: 137 ± 3 L-NAME: 157 ± 7, L-NAME + NO₂⁻: 136 ± 5 mm Hg). Also, NO₂⁻ infusion significantly increased VC when compared to L-NAME (P < 0.03), ultimately negating any significant differences from control animals (control: 0.78 ± 0.05, L-NAME: 0.57 ± 0.03, L-NAME + NO₂⁻; 0.69 ± 0.04 mL min⁻¹ 100 g⁻¹ mm Hg⁻¹) with no apparent fiber-type preferential effect. Overall, hindlimb BF was decreased significantly by L-NAME; however, in L-NAME + NO₂⁻, BF improved to a level not significantly different from healthy controls (control: 108 ± 8, L-NAME: 88 ± 3, L-NAME + NO₂⁻: 94 ± 6 mL min⁻¹ 100 g⁻¹, P = 0.38 L-NAME vs L-NAME + NO₂⁻). Individuals with diseases that impair NOS activity, and thus vascular function, may benefit from a NO₂⁻-based therapy in which NO bioavailability is elevated in an NOS-independent manner.

Keywords

nitric oxide nitrate blood flow vascular control

Introduction

The cardiovascular response to exercise is characterized by a multitude of neural, humoral, and mechanical components serving to elevate cardiac output and redistribute blood flow (BF), and thus O₂ delivery ( ${\dot{Q} O}_{2}$ ), to contracting myocytes. Of the humoral regulators, the ubiquitous signaling molecule nitric oxide (NO) plays a fundamental role in the hyperemic response to exercise, and, as a result, its bioavailability is key to elicit the changes in ${\dot{Q} O}_{2}$ necessary to meet the rapidly rising O₂ demand (oxygen uptake [ ${\dot{V} O}_{2}$ ]) of the skeletal muscle (reviewed by Joyner et al¹). Indeed, disease states hallmarked by reduced NO bioavailability (ie, chronic heart failure, CHF, reviewed by Poole et al²) demonstrate a robust disruption in spatial and temporal skeletal muscle ${\dot{Q} O}_{2}$ , resulting in perturbed metabolic function and compromised exercise tolerance.

NO is synthesized endogenously in a reaction catalyzed by the NO synthase (NOS) family of enzymes or the 1-step reduction of nitrite (NO₂⁻) to NO, the latter being a NOS-independent pathway (reviewed by Lundberg and Weitzberg³). Recent evidence from murine models suggests that the availability of NO₂⁻ may be upregulated via ingestion of nitrate (NO₃⁻)-rich food stuffs (ie, beetroot juice), thus elevating NO bioavailability (following the reduction of NO₃⁻ to NO₂⁻ and finally NO) resulting in improved skeletal muscle vascular, metabolic,^4
–6 and contractile⁷ function. These results extend to humans as several laboratories have demonstrated ergogenic effects of dietary NO₃⁻ supplementation in healthy^8

–13 and diseased^14

–17 populations. Interestingly, while these studies employ a dietary means of increasing endogenous [NO₂⁻], vasoactivity of the directly infused anion is evident in humans^18

–21 and animals,^22

–25 suggesting that bolus delivery may afford an expedited method of augmenting vascular and metabolic control in vivo.

Bearing in mind the beneficial impacts of dietary NO₃⁻ supplementation on exercise performance, and the vascular effects of NO₂⁻ infusion highlighted earlier, it is logical to consider that direct infusion with NO₂⁻ may also impact skeletal muscle vascular control during exercise. Furthermore, when considering that NO₂⁻ reduction to NO is potentiated in low PO₂ and/or pH environments,¹⁸ bioactivity of NO₂⁻ may be further facilitated (or relied upon) when NOS function is reduced or completely abolished and O₂ transport is impaired (as is the case in many pathological conditions). If direct NO₂⁻ infusion augments exercising skeletal muscle vascular function independent of NOS, NO₂⁻ therapy could emerge as an attractive means of restoring NO bioavailability in various cardiovascular diseases in which NOS function is compromised.

Despite these prospects, there are no investigations into the effects of NO₂⁻ infusion on exercising skeletal muscle vascular control under conditions of NOS blockade. Therefore, the purpose of this investigation was to determine the impacts of NO₂⁻ infusion on skeletal muscle vascular control during exercise in rats with NOS blockade elicited via L-NAME. We tested the hypothesis that, relative to the L-NAME condition, treatment with NO₂⁻ would restore exercising mean arterial pressure (MAP) and total exercising hindlimb skeletal muscle BF and vascular conductance (VC) to values observed in healthy young adult rats (with intact NOS function).

Methods

Ethical Approval

All procedures employed in this investigation were approved by the Institutional Animal Care and Use Committee of Kansas State University and were conducted under the guidelines established by The Journal of Physiology.²⁶ A total of 16 young adult male Sprague-Dawley rats (~3 months of age, Charles River Laboratories, Wilmington, Massachusetts) were maintained at accredited animal facilities at Kansas State University on a 12:12-hour light–dark cycle with food and water provided ad libitum. All rats were familiarized with running on a custom-built motor-driven treadmill for 5 min day⁻¹ at a speed of 20 m min⁻¹ up a 5% grade for ~5 days. In an effort to minimize the unnecessary use of additional animals, control BF, VC, blood gas, [lactate], and plasma [NO₂⁻]/[NO₃⁻] values reported herein represent animals from recently published work (n = 11,²⁷) and followed the same experimental procedures as detailed subsequently.

Surgical Instrumentation

On the day of the experiment, rats were anesthetized initially with a 5% isoflurane-O₂ mixture and maintained subsequently on 3% isoflurane/O₂ mixture. A catheter (PE-10 connected to PE-50, Intra-Medic polyethylene tubing, Clay Adams Brand, Becton, Dickinson and Company, Sparks, Maryland) was placed in the ascending aorta via the right carotid artery. A second catheter was surgically placed in the caudal (tail) artery as described previously.²⁸ Both catheters were tunneled subcutaneously through the dorsal aspect of the cervical region and exteriorized via a puncture wound in the skin. The incisions were closed, anesthesia was terminated, and the rats were given a minimum of 60 minutes to recover.²⁹

L-NAME Infusion

Rats were then placed on the treadmill and, following a ~5-minute resting period, N^G-nitro-L arginine methyl ester (10 mg kg⁻¹, L-NAME; n = 8, Sigma Chemical, St Louis, Missouri) was administered to each rat via the caudal artery catheter to inhibit NOS. This dose has been used extensively in our laboratory and has demonstrated inhibition of NOS via attenuation of acetylcholine-induced reductions in MAP.^30,31

Exercise Protocol and Measurement of Hindlimb Skeletal Muscle BF

Following L-NAME infusion, the caudal artery catheter was connected to a 1-mL syringe chambered in a Harvard infusion/withdrawal pump (model 907, Cambridge, Massachusetts) and the carotid artery catheter was connected to a pressure transducer (Gould Statham P23ID, Valley View, OH, USA) maintained at the same height as the animal. Approximately, 3 minutes post-L-NAME infusion, exercise was initiated and treadmill speed was increased progressively over a ~30-second period to a speed of 20 m min⁻¹ (5% grade, ~60% ${\dot{V} O}_{2}$ max³²). The rats continued to exercise for another 2.5 minutes until a total time of 3 minutes was reached. At 3 minutes, the Harvard pump was activated and withdrawal was initiated at a rate of 0.25 mL min⁻¹. Simultaneously, heart rate (HR) and MAP were measured and recorded. The carotid artery catheter was then disconnected from the pressure transducer and 0.5-0.6 × 10⁶ 15-μm diameter radiolabeled microspheres (⁵⁷Co or ⁸⁵Sr in random order; Perkin Elmer, Waltham, Massachusetts) were infused into the aortic arch for determination of regional BF (L-NAME condition). Following the microsphere infusion, ~0.3 mL of blood was sampled from the carotid artery catheter for the determination of blood [lactate] (Nova Stat Profile M, Nova Biomedical, Waltham, Massachusetts) and exercise was terminated.

NO₂⁻ Infusion

Following a 30-minute recovery period, a bolus infusion of sodium NO₂⁻ (7 mg kg⁻ ¹ body mass, L-NAME + NO₂⁻; n = 8, Sigma Chemical, St Louis, Missouri) was administered to each rat via the caudal artery catheter. The exercise and microsphere infusion protocols (radio-labeled differently from the first) were then repeated (condition L-NAME + NO₂⁻).

Blood Sampling and Measurement of Plasma [NO₃⁻] and [NO₂⁻]

Immediately following microsphere infusion but prior to the termination of exercise, a ~0.3-mL blood sample was drawn from the carotid artery catheter for determination of blood pH, PO₂, and % O₂ saturation (Nova Stat Profile M, Nova Biomedical,). For plasma [NO₃⁻] and [NO₂⁻], following the termination of exercise ~0.8 mL of blood was drawn into heparinized tubes and rapidly centrifuged at 5000g at 4°C for 6 minutes. Plasma was then extracted and frozen immediately at −80°C for later analysis via chemiluminescence as described previously.^4,5,27,33

Determination of BF and VC

Rats were euthanized via pentobarbital sodium overdose (≥50 mg kg⁻¹). The thorax of each rat was opened and accurate placement of the carotid artery catheter was confirmed before the internal organs and 28 individual muscles and muscle parts of the hindlimb were excised.

Radioactivity of each tissue was determined with a gamma scintillation counter (Packard Auto Gamma Spectrometer, model 5230, Downers Grove, Illinois). Tissue BF was then calculated using the reference sample method²⁸ and expressed as mL min⁻¹ 100g⁻¹. VC was then calculated by normalizing BF to MAP and expressed as mL· min⁻¹ 100g⁻¹ mm Hg⁻¹.

Statistical Analysis

Results were compared among (control vs L-NAME and control vs L-NAME + NO₂⁻) and within (L-NAME vs L-NAME + NO₂⁻) groups using a priori unpaired and paired 1-tail Student t tests, respectively, corrected for multiple comparisons. Values are expressed as mean ± standard error of the mean.

Results

Mean Arterial Pressure, HR, Plasma [NO₃⁻] and [NO₂⁻], and Blood Gases

Relative to control, post NO₂⁻ infusion plasma [NO₂⁻] (control: 0.17 ± 0.2, L-NAME + NO₂⁻: 306.8 ± 38.7 µmol/L, P < 0.01) and [NO₃⁻] (control: 17.8 ± 1, L-NAME + NO₂⁻: 152.5 ± 35 µmol/L, P < 0.01) were significantly elevated. Relative to control, MAP was significantly higher in the L-NAME condition (Figure 1, P < 0.03). Following NO₂⁻ infusion, MAP was reduced significantly when compared to the L-NAME condition (P < 0.03). Exercising MAP was not different between control and L-NAME + NO₂⁻ groups (P = 0.36). Relative to the control and L-NAME + NO₂⁻ conditions, exercising HR was significantly lower in the L-NAME condition (control: 528 ± 12, L-NAME: 493 ± 37, L-NAME + NO₂⁻: 520 ± 33 beats min⁻¹, P < 0.01).

Figure 1.

Exercising mean arterial pressure (MAP), systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure (PP) values for control, L-NAME, and L-NAME + NO₂⁻ conditions. *P < 0.03 vs control, #P < 0.03 vs L-NAME. Note: control values represented are from previously published data.

There were no differences in arterial PO₂, PCO₂, or % O₂ saturation during exercise. Arterial blood [lactate] during exercise was greater following NO₂⁻ infusion (3.8 ± 0.5 mmol/L) compared to control (2.7 ± 0.4 mmol/L) and L-NAME only (2.1 ± 0.3 mmol/L) conditions, (P < 0.016).

BF and VC

L-NAME significantly reduced exercising total hindlimb skeletal muscle BF and VC (Figure 2, P < 0.03). Following NO₂⁻ infusion, total hindlimb skeletal muscle VC was restored to levels observed in control rats (Figure 2, P < 0.03 L-NAME vs L-NAME+NO₂⁻, P > 0.10 control vs L-NAME + NO₂). There were no differences in total hindlimb skeletal muscle BF during exercise in L-NAME vs L-NAME + NO₂⁻ or control versus L-NAME + NO₂⁻ conditions (Figure 2 bottom panel, P > 0.03).

Figure 2.

Total hindlimb skeletal muscle blood flow (BF) and vascular conductance (VC) for control, L-NAME, and L-NAME + NO₂⁻ conditions in rats during submaximal locomotory exercise. *P < 0.03 vs control, #P < 0.03 vs L-NAME. Note: control values represented are from previously published data.

Relative to control, L-NAME-treated rats had lower BF in 5 and VC in 15 of the 28 individual hindlimb muscles and muscle parts, whereas this was the case for only 3 muscles (BF and VC) in the L-NAME+NO₂⁻ condition (Table 1, P < 0.03 for all). Moreover, following NO₂⁻ infusion, VC in 19 of the 28 individual hindlimb muscles and muscle parts was increased significantly when compared to the L-NAME condition (P < 0.03, Table 1).

Table 1.

Effects NO₂⁻ Infusion (7 mg kg⁻¹) on Exercising Hindlimb Skeletal Muscle BF (mL min⁻¹ 100g⁻¹) and VC (mL min⁻¹ 100g⁻¹ mm Hg⁻¹) in Rats With NOS Blockade (L-NAME).^a

	BF			VC
	Control	L-NAME	L-NAME+NO₂⁻	Control	L-NAME	L-NAME+NO₂⁻
Ankle extensors
Soleus (9%)	295 ± 42	242 ± 71	285 ± 36	2.14 ± 0.30	1.56 ± 0.17	2.06 ± 0.23^b
Plantaris (80%)	207 ± 15	144 ± 8^c	173 ± 15	1.50 ± 0.10	0.93 ± 0.06^c	1.27 ± 0.08^b
Gastrocnemius, red (14%)	452 ± 44	333 ± 59	362 ± 65	3.27 ± 0.30	2.18 ± 0.02^c	2.63 ± 0.44^b
Gastrocnemius, white (100%)	42 ± 7	26 ± 3	37 ± 4…	0.30 ± 0.05	0.17 ± 0.02^c	0.27 ± 0.03^b
Gastrocnemius, mixed (91%)	149 ± 12	120 ± 5	141 ± 8	1.08 ± 0.08	0.77 ± 0.04^c	1.04 ± 0.04^b
Tibialis posterior (73%)	118 ± 17	81 ± 12	91 ± 13	0.85 ± 0.12	0.51 ± 0.07^c	0.66 ± 0.09^b
Flexor digitorum longus (68%)	99 ± 14	60 ± 7^c	69 ± 9	0.71 ± 0.09	0.38 ± 0.04^c	0.51 ± 0.06^b
Flexor halicus longus (71%)	75 ± 10	68 ± 8	99 ± 14^b	0.54 ± 0.06	0.44 ± 0.06	0.74 ± 0.11^b
Ankle flexors
Tibialis anterior, red (63%)	343 ± 35	209 ± 10^c	219 ± 20^c	2.48 ± 0.23	1.36 ± 0.10^c	1.62 ± 0.14^c
Tibialis anterior, white (80%)	119 ± 14	83 ± 6^c	89 ± 12	0.86 ± 0.09	0.54 ± 0.05^c	0.66 ± 0.09^b
Extensor digitorum longus (76%)	54 ± 7	75 ± 20	77 ± 17	0.39 ± 0.05	0.50 ± 0.14	0.57 ± 0.13^b
Peroneals (67%)	128 ± 11	72 ± 14^c	91 ± 13^c	0.93 ± 0.08	0.46 ± 0.09^c	0.67 ± 0.09^b,c
Knee extensors
Vastus intermedius (4%)	359 ± 39	257 ± 25	302 ± 39	2.60 ± 0.27	1.66 ± 0.17^c	2.20 ± 0.25^b
Vastus medialis (82%)	114 ± 18	137 ± 13	144 ± 14	0.82 ± 0.12	0.89 ± 0.08	1.06 ± 0.08^b
Vastus lateralis, red (35%)	388 ± 43	310 ± 35	281 ± 25	2.82 ± 0.29	2.02 ± 0.26	2.08 ± 0.52
Vastus lateralis, white (100%)	33 ± 5	26 ± 8	31 ± 7	0.24 ± 0.03	0.16 ± 0.04	0.23 ± 0.04^b
Vastus lateralis, mixed (89%)	167 ± 21	123 ± 12	127 ± 13	1.22 ± 0.14	0.81 ± 0.09^c	0.94 ± 0.09^b
Rectus femoris, red (66%)	224 ± 33	181 ± 15	204 ± 17	1.62 ± 0.23	1.17 ± 0.10	1.50 ± 0.11^b
Rectus femoris, white (100%)	101 ± 13	81 ± 7	91 ± 8	0.73 ± 0.09	0.52 ± 0.05	0.67 ± 0.06^b
Knee flexors
Biceps femoris anterior (100%)	50 ± 8	33 ± 4	36 ± 4	0.36 ± 0.05	0.21 ± 0.03^c	0.27 ± 0.03^b
Biceps femoris posterior (92%)	79 ± 8	65 ± 3	71 ± 5	0.58 ± 0.06	0.42 ± 0.02^c	0.53 ± 0.04^b
Semitendinosus (83%)	56 ± 6	34 ± 3^c	37 ± 4^c	0.40 ± 0.04	0.22 ± 0.02^c	0.28 ± 0.03^c
Semimembranosus, red (72%)	119 ± 14	86 ± 7	83 ± 14	0.87 ± 0.09	0.56 ± 0.05^c	0.62 ± 0.11
Semimembranosus, white (100%)	33 ± 6	38 ± 7	40 ± 11	0.24 ± 0.04	0.25 ± 0.05	0.30 ± 0.09
Thigh Adductors
Adductor longus (5%)	315 ± 38	263 ± 26	231 ± 31^b	2.28 ± 0.26	1.71 ± 0.21	1.68 ± 0.22
Adductor magnus & brevis (89%)	83 ± 8	80 ± 7	80 ± 9	0.60 ± 0.05	0.52 ± 0.05	0.60 ± 0.06
Gracilis (77%)	42 ± 4	37 ± 4	34 ± 5	0.30 ± 0.03	0.24 ± 0.02	0.26 ± 0.04
Pectineus (69%)	54 ± 8	40 ± 6	46 ± 11	0.39 ± 0.06	0.25 ± 0.03	0.34 ± 0.08

Abbreviations: BF, blood flow; NOS, nitric oxide synthase; SEM, standard error of the mean;VC, vascular conductance.

^aData are mean ± SEM. Values in parentheses indicate % type IIb + d/x according to Delp & Duan⁵². Control: n = 11, L-NAME: n = 8, L-NAME + NO₂⁻: n = 8.

^bP < .03 vs L-NAME.

^cP < .03 vs control.

Relative to control, BF and VC were lower in the adrenals and pancreas while VC was lower in the kidneys, stomach, and small intestine in rats treated with L-NAME (P < .03, Table 2). Following NO₂⁻ infusion, renal and adrenal BF and VC were lower when compared to control animals, while renal and adrenal BF was reduced when compared to L-NAME (P < .03, Table 2).

Table 2.

Effects of NO₂⁻ Infusion (7 mg kg⁻¹) on Exercising BF (mL min⁻¹ 100g⁻¹) and VC (mL ·min⁻¹ 100g⁻ ¹ mm Hg⁻ ¹) in the Kidneys and Organs of the Splanchnic Region.^a

	BF			VC
	Control	L-NAME	L-NAME + NO₂	Control	L-NAME	L-NAME + NO₂
Kidney	421 ± 42	338 ± 28	267 ± 31^b,c	3.05 ± 0.28	2.22 ± 0.25^b	1.96 ± 0.22^b
Stomach	67 ± 13	38 ± 3	35 ± 4	0.49 ± 0.10	0.25 ± 0.02^b	0.25 ± 0.03
Adrenals	353 ± 72	128 ± 17^c	100 ± 66^b	2.87 ± 0.44	0.85 ± 0.14^b	0.72 ± 0.15^b
Spleen	61 ± 14	102 ± 21	48 ± 7^c	0.44 ± 0.10	0.68 ± 0.16	0.35 ± 0.06
Pancreas	110 ± 15	72 ± 8^b	93 ± 22	0.80 ± 0.11	0.47 ± 0.06^b	0.67 ± 0.15
Small intestine	240 ± 27	177 ± 24	211 ± 26	1.74 ± 0.18	1.17 ± 0.19^b	1.55 ± 0.17
Large intestine	127 ± 16	123 ± 20	140 ± 42	0.92 ± 0.10	0.82 ± 0.15	1.01 ± 0.28
Liver^d	16 ± 4	15 ± 2	13 ± 3	0.12 ± 0.02	0.10 ± 0.01	0.09 ± 0.02

Abbreviations: BF, blood flow; SEM, standard error of the mean; VC, vascular conductance.

^aData are mean ± SEM. Control: n = 11, L-NAME: n = 8, L-NAME + NO₂⁻: n = 8.

^bP < .03 vs control.

^cP < .03 vs L-NAME.

^dindicates arterial, not portal, BF and VC.

Discussion

The principal original finding of this investigation is that, in the face of NOS blockade, NO₂⁻ infusion restored exercising MAP and hindlimb skeletal muscle VC to levels observed in young adult healthy rats with intact NOS function. While NO₂⁻ infusion did not increase BF when compared to the L-NAME condition, it did abolish the lower BF induced by L-NAME. Elevations in VC and reductions in MAP could serve to reduce afterload and thus reduce the work of the heart during exercise. These results demonstrate that NO₂⁻ may serve as a powerful modulator of vascular control in vivo, independent of NOS function and thus may hold promising therapeutic potential, particularly in diseases with impaired NOS function and chronically elevated MAP.

Effects of Inorganic NO₂⁻ Infusion on Skeletal Muscle BF and VC and MAP

An abundance of research has focused on defining the vasoactive/cardioprotective roles of NO₂⁻ with many studies suggesting that the reduction of NO₂⁻ to NO compliments the well-understood NOS pathway of NO production, particularly when NOS function becomes uncoupled or otherwise impaired (reviewed by^34,35). The vascular responses to NO₂⁻ infusion presented herein support this notion. Similar to what has been reported previously in our laboratory,^36,37 infusion with the comprehensive NOS blocker L-NAME increased MAP ~15% and decreased skeletal muscle VC ~26% during exercise. Consistent with our hypothesis, infusion with NO₂⁻ (7 mg kg⁻¹) restored MAP and VC to levels similar to those observed in healthy control animals. One potential explanation for these effects of NO₂⁻ could be the lower PO₂/pH environment present within the skeletal muscle following NOS inhibition.³³ Such environments facilitate (or uninhibit) NO₂⁻ reduction to NO in vivo,^18,38 which may allow local NO₂⁻ to support the blood–myocyte Po₂ gradient (via ↑ ${\dot{Q} O}_{2}$ and microvascular Po ₂, Po ₂mv) that, when compromised, leads to tissue hypoxia and exacerbates intracellular metabolic perturbations.³⁹

One striking aspect of this investigation, in which acute NO₂⁻ infusion was employed, was that the augmented skeletal muscle VC was observed in muscles and muscle parts that span the full spectrum of fast- and slow-twitch fiber types (Table 1). This is in contrast to investigations using short-term dietary NO₃⁻ supplementation as a means of increasing circulating [NO₂⁻]. Specifically, there is a fiber-type preferential effect of dietary NO₃⁻ supplementation as rats given NO₃⁻-rich beetroot juice for 5 days exhibited elevated skeletal muscle BF and VC exclusively in muscles and muscle portions comprised of ≥ 66% type IIb + d/x muscle fibres.²⁷ Accordingly, beetroot juice elevates PO₂mv during muscle contractions in the gastrocnemius (fast-twitch) but not soleus (slow-twitch) muscles.³³ The substantial array of muscles and muscle portions exhibiting a vasoactive response to NO₂⁻ infusion herein suggests that the fiber-type preferential effects observed following dietary NO₃⁻ supplementation may be conferred via changes in protein expression that require a longer period of elevated NO₂⁻ exposure to manifest. This idea is supported by evidence from Hernandez et al⁷ in which the improvements in fast-twitch skeletal muscle force production evoked by NO₃⁻ supplementation were attributed to elevations in calcium handling proteins (ie, calsequestrin 1 and the dihydropyridine receptor), which were present following multiple days of dietary NO₃⁻ supplementation.

Additionally, the discrepancies in the vascular responses to NO₃⁻ versus NO₂⁻ treatment could be related to the relative impacts of NOS inhibition in fast- versus slow-twitch muscles. Skeletal muscles comprised predominantly of slow-twitch fibers demonstrate the greatest deficits in BF and $\dot{V} C$ following L-NAME infusion³⁶ likely due to a greater expression of endothelial NOS (eNOS) within these tissues.⁴⁰ These slow-twitch muscles may exhibit much greater BF and ${\dot{V} O}_{2}$ than their fast-twitch counterparts both at rest and during exercise (~100% greater for both BF and ${\dot{V} O}_{2}$ ⁴¹). Consequently, NOS inhibition may have crippled O₂ delivery in these muscles sufficiently enough to produce an environment ripe for NO₂⁻ bioactivation (ie, very low PO₂ and pH). This effect could place more emphasis on NO₂⁻, as the primary source of NO in these specific tissues when vascular function is impaired as it is in many disease states.⁴² In this regard, the spatial changes in VC seen following NO₂⁻ infusion herein may mimic closely what would be observed in individuals with diseases that compromise NOS function. However, these questions require further investigation using specific models of vascular disease.

Clinical and Therapeutic Implications

In healthy individuals, eNOS is the primary endogenous source for NO₂⁻ and NO.⁴³ Endothelial dysfunction becomes evident early on in many diseases, including CHF (reviewed by Poole et al²) and peripheral artery disease (reviewed by Brevetti et al⁴⁴) and thus likely limits vascular and metabolic function via attenuated NO production from both NOS-dependent and -independent pathways.^43,45 As evidenced by Hirai et al,^46,47 reduced NO from NOS dramatically impairs the matching of skeletal muscle ${\dot{Q} O}_{2}$ to ${\dot{V} O}_{2}$ such that superfusion of L-NAME in the contracting rat spinotrapezius muscle transforms the healthy PO₂mv profile into one resembling CHF.⁴⁶ In this regard, the blockade of NOS induced by L-NAME infusion performed in the present investigation presents a challenge that mimics the consequences of CHF and potentially other diseases. Therefore, from the present findings, a therapy in which systemic [NO₂⁻] is elevated (via endogenous or exogenous sources) may provide beneficial vascular responses independent of NOS function. Even small improvements in vascular function may enhance metabolic control during dynamic exercise, potentially improving adherence to rehabilitation programs³⁵ which in-and-of themselves would upregulate eNOS function and endogenous NO₂⁻ production.

Experimental Considerations and Potential Limitations

A surprising result of the present investigation was the rise in exercising blood [lactate] following NO₂⁻ infusion (~41% and 81% greater vs control and L-NAME, respectively). Lower levels of NO may act as a useful brake on mitochondrial activity via competitive binding to complex IV of the respiratory chain.⁴⁸ In contrast, high concentrations of NO have been associated with adverse effects on cell respiration via nitrosylation of mitochondrial electron chain complexes, specifically complex I.⁴⁹ In addition, NO works to inhibit complex IV (cytochrome oxidase), thereby reducing cellular O₂ consumption. Both of these effects may prove beneficial in certain environments or situations when O₂ delivery becomes reduced as reductions in tissue ${\dot{V} O}_{2}$ work to elevate the intramyocyte PO₂ gradient across a larger tissue area, effectively sharing the available O₂.⁵⁰ However, in the current study, it is possible that the rate of NO₂⁻ reduction to NO became high enough to overwhelm mitochondrial respiration, thus leading to impaired oxidative metabolism and an increased reliance on glycolytic means of ATP production. In addition, while the current dose of NO₂⁻raised plasma [NO₃⁻] to levels very similar to what has been reported following dietary NO₃⁻ supplementation in humans^9,14 and animals^5,27 the plasma [NO₂⁻] were much greater than that achieved via NO₃⁻ supplementation and thus may have contributed to the aforementioned effect on metabolism. In this regard, a comprehensive dose–response relationship will need to be determined before NO₂⁻ can be used as an effective therapeutic.

Furthermore, considering that NOS was acutely inhibited in the present investigation, the impacts of NO₂⁻ infusion may differ when administered to specific models of vascular diseases that have been developed chronically, as this would more closely mimic specific etiologies. Additionally, due to the relatively long half-life and bioactivity of L-NAME metabolites (~20 hours in rats⁵¹) the experimental design was limited to a fixed sequence and therefore, an ordering effect cannot be ruled out. Future investigations in which NO₂⁻ is employed in healthy control animals would also provide further insight into the bioactivity of NO₂⁻ in animals with intact NOS function and could shed light on how a NO₂⁻-based intervention may impact healthy cardiovascular function.

Conclusion

These data highlight the potential for NO₂⁻ to act independently of NOS and improve skeletal muscle vascular control during exercise. Considering the multiple cardiovascular diseases that impair NOS function, therapies that increase [NO₂⁻] may result in improved skeletal muscle vascular control during exercise. However, the NO₂⁻ induced changes in blood [lactate] seen during exercise herein suggests that the reduction of NO₃⁻ to NO₂⁻, accomplished via facultative anaerobes in the mouth following dietary NO₃⁻ consumption, may provide the controlled release of NO₂⁻ needed to elicit the most beneficial vascular and metabolic changes during exercise. It is anticipated that future investigations into the vascular impacts of both NO₂⁻- and NO₃⁻-based therapies will provide crucial insight into the potential benefits, and limitations, of both interventions.

Footnotes

Author Contributions

SKF, CTH, AMJ, TIM, and DCP contributed to conception and design of the experiments.

SKF, AAG, CTH, JLW, AJF, TDC, TS, JDA, AMJ, TIM, and DCP contributed to collection, analysis, and interpretation of data. SKF, CTH, TDC, JDA, AMJ, TIM, and DCP contributed to drafting the article and revising it critically for important intellectual content. All the authors have approved the final version of the article.

Acknowledgments

The authors would like to thank Ms K. Sue Hageman for her excellent technical assistance.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: These experiments were funded by a Kansas State University SMILE award to TIM, and American Heart Association Midwest Affiliate (10GRNT4350011) and NIH (HL-108328) awards to DCP.

References

Joyner

Tschakovsky

. Nitric oxide and physiologic vasodilation in human limbs: where do we go from here? Can J Appl Physiol. 2003;28(3):475–490.

Poole

Hirai

Copp

Musch

. Muscle oxygen transport and utilization in heart failure: implications for exercise (in)tolerance. Am J Physiol Heart Circ Physiol. 2012;302(5):H1050–H1063.

Lundberg

Weitzberg

. NO generation from inorganic nitrate and nitrite: role in physiology, nutrition and therapeutics. Arch Pharm Res. 2009;32(8):1119–1126.

Ferguson

Hirai

Copp

. Effects of nitrate supplementation via beetroot juice on contracting rat skeletal muscle microvascular oxygen pressure dynamics. Respir Physiol Neurobiol. 2013;187(3):250–255.

Ferguson

Hirai

Copp

. Dose dependent effects of nitrate supplementation on cardiovascular control and microvascular oxygenation dynamics in healthy rats. Nitric Oxide. 2014;39:51–58.

Larsen

Schiffer

Borniquel

. Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab. 2011;13(2):149–159.

Hernandez

Schiffer

Ivarsson

. Dietary nitrate increases tetanic [Ca²⁺]i and contractile force in mouse fast-twitch muscle. J Physiol. 2012;590(pt 15):3575–3583.

Bailey

Fulford

Vanhatalo

. Dietary nitrate supplementation enhances muscle contractile efficiency during knee-extensor exercise in humans. J Appl Physiol. 2010;109(1):135–148.

Bailey

Winyard

Vanhatalo

. Dietary nitrate supplementation reduces the O₂ cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol. 2009;107(4):1144–1155.

10.

Muggeridge

Howe

Spendiff

Pedlar

James

Easton

. The effects of a single dose of concentrated beetroot juice on performance in trained flatwater kayakers. Int J Sport Nutr Exerc Metab. 2013;23(5):498–506.

11.

Vanhatalo

Bailey

Blackwell

. Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise. Am J Physiol Regul Integr Comp Physiol. 2010;299(4):R1121–R1131.

12.

Vanhatalo

Fulford

Bailey

Blackwell

Winyard

Jones

. Dietary nitrate reduces muscle metabolic perturbation and improves exercise tolerance in hypoxia. J Physiol. 2011;589(pt 22):5517–5528.

13.

Wylie

Mohr

Krustrup

. Dietary nitrate supplementation improves team sport-specific intense intermittent exercise performance. Eur J Appl Physiol. 2013;113(7):1673–1684.

14.

Kenjale

Ham

Stabler

. Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease. J Appl Physiol. 2011;110(6):1582–1591.

15.

Allen

Stabler

Kenjale

. Plasma nitrite flux predicts exercise performance in peripheral arterial disease after 3 months of exercise training. Free Radic Biol Med. 2010;49(6):1138–1144.

16.

Berry

Justus

Hauser

. Dietary nitrate supplementation improves exercise performance and decreases blood pressure in COPD patients. Nitric Oxide. 2015;48:22–30.

17.

Zamani

Rawat

Shiva-Kumar

. The effect of inorganic nitrate on exercise capacity in heart failure with preserved ejection fraction. Circulation. 2015;131(4):371–380.

18.

Cosby

Partovi

Crawford

. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003;9(12):1498–1505.

19.

Ingram

Pinder

Bailey

Fraser

James

. Low-dose sodium nitrite vasodilates hypoxic human pulmonary vasculature by a means that is not dependent on a simultaneous elevation in plasma nitrite. Am J Physiol Heart Circ Physiol. 2010;298(2):H331–H339.

20.

Maher

Arif

Madhani

. Impact of chronic congestive heart failure on pharmacokinetics and vasomotor effects of infused nitrite. Br J Pharmacol. 2013;169(3):659–670.

21.

Pluta

Oldfield

Bakhtian

. Safety and feasibility of long-term intravenous sodium nitrite infusion in healthy volunteers. PloS One. 2011;6(1):e14504.

22.

Alzawahra

Talukder

Liu

Samouilov

Zweier

. Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall. Am J Physiol Heart Circ Physiol. 2008;295(2):H499–H508.

23.

Ghosh

Kapil

Fuentes-Calvo

. Enhanced vasodilator activity of nitrite in hypertension: critical role for erythrocytic xanthine oxidoreductase and translational potential. Hypertension. 2013;61(5):1091–1102.

24.

Pinheiro

Montenegro

Amaral

Ferreira

Oliveira

Tanus-Santos

. Increase in gastric pH reduces hypotensive effect of oral sodium nitrite in rats. Free Radic Biol Med. 2012;53(4):701–709.

25.

Sindler

Fleenor

Calvert

. Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging. Aging Cell. 2011;10(3):429–437.

26.

Drummond

. Reporting ethical matters in the Journal of Physiology: standards and advice. J Physiol. 2009;587(pt 4):713–719.

27.

Ferguson

Hirai

Copp

. Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control in rats. J Physiol. 2013;591(pt 2):547–557.

28.

Musch

Terrell

. Skeletal muscle blood flow abnormalities in rats with a chronic myocardial infarction: rest and exercise. Am J Physiol. 1992;262(2 pt 2):H411–H419.

29.

Flaim

Nellis

Toggart

Drexler

Kanda

Newman

. Multiple simultaneous determinations of hemodynamics and flow distribution in conscious rat. J Pharmacol Meth. 1984;11(1):1–39.

30.

Copp

Hirai

Hageman

Poole

Musch

. Nitric oxide synthase inhibition during treadmill exercise reveals fiber-type specific vascular control in the rat hindlimb. Am J Physiol Regul Integr Comp Physiol. 2010;298(2):R478–R485.

31.

Hirai

Copp

Hageman

Poole

Musch

. Aging alters the contribution of nitric oxide to regional muscle hemodynamic control at rest and during exercise in rats. J Appl Physiol. 2011;111(4):989–998.

32.

Musch

Bruno

Bradford

Vayonis

Moore

. Measurements of metabolic rate in rats: a comparison of techniques. J Appl Physiol. 1988;65(2):964–970.

33.

Ferguson

Holdsworth

Wright

. Microvascular oxygen pressures in muscles comprised of different fiber types: Impact of dietary nitrate supplementation. Nitric Oxide. 2015;48:38–43.

34.

Bailey

Feelisch

Horowitz

Frenneaux

Madhani

. Pharmacology and therapeutic role of inorganic nitrite and nitrate in vasodilatation. Pharmacol Ther. 2014;144(3):303–320.

35.

Allen

Giordano

Kevil

. Nitrite and nitric oxide metabolism in peripheral artery disease. Nitric Oxide. 2012;26(4):217–222.

36.

Hirai

Visneski

Kearns

Zelis

Musch

. Effects of NO synthase inhibition on the muscular blood flow response to treadmill exercise in rats. J Appl Physiol. 1994;77(3):1288–1293.

37.

Musch

McAllister

Symons

. Effects of nitric oxide synthase inhibition on vascular conductance during high speed treadmill exercise in rats. Exp Physiol. 2001;86(6):749–757.

38.

Feelisch

Fernandez

Bryan

. Tissue processing of nitrite in hypoxia: an intricate interplay of nitric oxide-generating and -scavenging systems. J Biol Chem. 2008;283(49):33927–33934.

39.

Arthur

Hogan

Bebout

Wagner

Hochachka

. Modeling the effects of hypoxia on ATP turnover in exercising muscle. J Appl Physiol. 1992;73(2):737–742.

40.

Woodman

Schrage

Rush

. Hindlimb unweighting decreases endothelium-dependent dilation and eNOS expression in soleus not gastrocnemius. J Appl Physiol. 2001;91(3):1091–1098.

41.

Behnke

McDonough

Padilla

Musch

Poole

. Oxygen exchange profile in rat muscles of contrasting fibre types. J Physiol. 2003;549(pt 2):597–605.

42.

Behnke

Delp

McDonough

Spier

Poole

Musch

. Effects of chronic heart failure on microvascular oxygen exchange dynamics in muscles of contrasting fiber type. Cardiovasc Res. 2004;61(2):325–332.

43.

Kleinbongard

Dejam

Lauer

. Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic Biol Med. 2006;40(2):295–302.

44.

Brevetti

Silvestro

Schiano

Chiariello

. Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease: additive value of flow-mediated dilation to ankle-brachial pressure index. Circulation. 2003;108(17):2093–2098.

45.

Kleinbongard

Dejam

Lauer

. Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals. Free Radic Biol Med. 2003;35(7):790–796.

46.

Ferreira

Hageman

Hahn

. Muscle microvascular oxygenation in chronic heart failure: role of nitric oxide availability. Acta Physiol. 2006;188(1):3–13.

47.

Ferreira

Padilla

Williams

Hageman

Musch

Poole

. Effects of altered nitric oxide availability on rat muscle microvascular oxygenation during contractions. Acta physiol. 2006;186(3):223–232.

48.

Erusalimsky

Moncada

. Nitric oxide and mitochondrial signaling: from physiology to pathophysiology. Arterioscler Thromb Vasc Biol. 2007;27(12):2524–2531.

49.

Clementi

Brown

Feelisch

Moncada

. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci U S A. 1998;95(13):7631–7636.

50.

Thomas

Liu

Kantrow

Lancaster

The biological lifetime of nitric oxide: implications for the perivascular dynamics of NO and O₂. Proc Natl Acad Sci U S A. 2001;98(1):355–360.

51.

Vitecek

Lojek

Valacchi

Kubala

. Arginine-based inhibitors of nitric oxide synthase: therapeutic potential and challenges. Mediators Inflamm. 2012;2012:318087.

52.

Delp

Duan

. Composition and size of type I, IIA, IID/X, and IIB fibers and citrate synthase activity of rat muscle. Journal of applied physiology. 1996;80(1):261–270.

Skeletal Muscle Vascular Control During Exercise

Abstract

Keywords

Introduction

Methods

Ethical Approval

Surgical Instrumentation

L-NAME Infusion

Exercise Protocol and Measurement of Hindlimb Skeletal Muscle BF

NO2− Infusion

Blood Sampling and Measurement of Plasma [NO3−] and [NO2−]

Determination of BF and VC

Statistical Analysis

Results

Mean Arterial Pressure, HR, Plasma [NO3−] and [NO2−], and Blood Gases

BF and VC

Discussion

Effects of Inorganic NO2− Infusion on Skeletal Muscle BF and VC and MAP

Clinical and Therapeutic Implications

Experimental Considerations and Potential Limitations

Conclusion

Footnotes

Author Contributions

Acknowledgments

Declaration of Conflicting Interests

Funding

References

NO₂⁻ Infusion

Blood Sampling and Measurement of Plasma [NO₃⁻] and [NO₂⁻]

Mean Arterial Pressure, HR, Plasma [NO₃⁻] and [NO₂⁻], and Blood Gases

Effects of Inorganic NO₂⁻ Infusion on Skeletal Muscle BF and VC and MAP