Brad W. Wilkins

Nitric oxide contributes to the augmented vasodilatation during hypoxic exercise

We tested the hypotheses that (1) nitric oxide (NO) contributes to augmented skeletal muscle vasodilatation during hypoxic exercise and (2) the combined inhibition of NO production and adenosine receptor activation would attenuate the augmented vasodilatation during hypoxic exercise more than NO inhibition alone. In separate protocols subjects performed forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O2 saturation). In protocol 1 (n= 12), subjects received intra‐arterial administration of saline (control) and the NO synthase inhibitor NG‐monomethyl‐l‐arginine (l‐NMMA). In protocol 2 (n= 10), subjects received intra‐arterial saline (control) and combined l‐NMMA–aminophylline (adenosine receptor antagonist) administration. Forearm vascular conductance (FVC; ml min−1 (100 mmHg)−1) was calculated from forearm blood flow (ml min−1) and blood pressure (mmHg). In protocol 1, the change in FVC (Δ from normoxic baseline) due to hypoxia under resting conditions and during hypoxic exercise was substantially lower with l‐NMMA administration compared to saline (control; P < 0.01). In protocol 2, administration of combined l‐NMMA–aminophylline reduced the ΔFVC due to hypoxic exercise compared to saline (control; P < 0.01). However, the relative reduction in ΔFVC compared to the respective control (saline) conditions was similar between l‐NMMA only (protocol 1) and combined l‐NMMA–aminophylline (protocol 2) at 10% (−17.5 ± 3.7 vs.−21.4 ± 5.2%; P= 0.28) and 20% (−13.4 ± 3.5 vs.−18.8 ± 4.5%; P= 0.18) hypoxic exercise. These findings suggest that NO contributes to the augmented vasodilatation observed during hypoxic exercise independent of adenosine.

Exercise hyperaemia: Is anything obligatory but the hyperaemia?

Exercise can increase skeletal muscle blood flow by 100‐fold over values observed at rest. As this value was 3 to 4 times higher than so‐called ‘textbook’ values at the time it raised a number of issues about cardiovascular control. However, there is a continuing inability to identify the factor or combination of factors that explain this substantial increase in muscle blood flow. Moreover, these governing mechanism(s) must also explain the precise matching of muscle blood flow to metabolic demand and oxygen use or need. The difficulties identifying the mechanisms for exercise hyperaemia are especially disappointing due to the essentially concurrent discovery in the 1980s that the vascular endothelium was a key site of vasomotor control and that nitric oxide (NO) potentially released from nerves, endothelial cells, directly from tissues such as skeletal muscle, or perhaps released from red blood cells, might participate in vascular control in a way that would permit blood flow and metabolism to be closely matched.

H1 receptor‐mediated vasodilatation contributes to postexercise hypotension

In normally active individuals, postexercise hypotension after a single bout of aerobic exercise is due to an unexplained peripheral vasodilatation. Histamine has been shown to be released during exercise and could contribute to postexercise vasodilatation via H1 receptors in the peripheral vasculature. The purpose of this study was to determine the potential contribution of an H1 receptor‐mediated vasodilatation to postexercise hypotension. We studied 14 healthy normotensive men and women (ages 21.9 ± 2.1 years) before and through to 90 min after a 60 min bout of cycling at 60% on randomized control and H1 receptor antagonist days (540 mg oral fexofenadine hydrochloride; Allegra). Arterial blood pressure (automated auscultation) and femoral blood flow (Doppler ultrasound) were measured in the supine position. Femoral vascular conductance was calculated as flow/pressure. Fexofenadine had no effect on pre‐exercise femoral vascular conductance or mean arterial pressure (P > 0.5). At 30 min postexercise on the control day, femoral vascular conductance was increased (Δ+33.7 ± 7.8%; P < 0.05 versus pre‐exercise) while mean arterial pressure was reduced (Δ−6.5 ± 1.6 mmHg; P < 0.05 versus pre‐exercise). In contrast, at 30 min postexercise on the fexofenadine day, femoral vascular conductance was not elevated (Δ+10.7 ± 9.8%; P= 0.7 versus pre‐exercise) and mean arterial pressure was not reduced (Δ−1.7 ± 1.2 mmHg; P= 0.2 versus pre‐exercise). Thus, ingestion of an H1 receptor antagonist markedly reduces vasodilatation after exercise and blunts postexercise hypotension. These data suggest H1 receptor‐mediated vasodilatation contributes to postexercise hypotension.

Exercise intensity-dependent contribution of β-adrenergic receptor-mediated vasodilatation in hypoxic humans

We previously reported that hypoxia‐mediated reductions in α‐adrenoceptor sensitivity do not explain the augmented vasodilatation during hypoxic exercise, suggesting an enhanced vasodilator signal. We hypothesized that β‐adrenoceptor activation contributes to augmented hypoxic exercise vasodilatation. Fourteen subjects (age: 29 ± 2 years) breathed hypoxic gas to titrate arterial O2 saturation (pulse oximetry) to 80%, while remaining normocapnic via a rebreath system. Brachial artery and antecubital vein catheters were placed in the exercising arm. Under normoxic and hypoxic conditions, baseline and incremental forearm exercise (10% and 20% of maximum) was performed during control (saline), α‐adrenoceptor inhibition (phentolamine), and combined α‐ and β‐adrenoceptor inhibition (phentolomine/propranolol). Forearm blood flow (FBF), heart rate, blood pressure, minute ventilation, and end‐tidal CO2 were determined. Hypoxia increased heart rate (P < 0.05) and minute ventilation (P < 0.05) at rest and exercise under all drug infusions, whereas mean arterial pressure was unchanged. Arterial adrenaline (P < 0.05) and venous noradrenaline (P < 0.05) were higher with hypoxia during all drug infusions. The change (Δ) in FBF during 10% hypoxic exercise was greater with phentolamine (Δ306 ± 43 ml min−1) vs. saline (Δ169 ± 30 ml min−1) or combined phentolamine/propranolol (Δ213 ± 25 ml min−1; P < 0.05 for both). During 20% hypoxic exercise, ΔFBF was greater with phentalomine (Δ466 ± 57 ml min−1; P < 0.05) vs. saline (Δ346 ± 40 ml min−1) but was similar to combined phentolamine/propranolol (Δ450 ± 43 ml min−1). Thus, in the absence of overlying vasoconstriction, the contribution of β‐adrenergic mechanisms to the augmented hypoxic vasodilatation is dependent on exercise intensity.

Nitric oxide is not permissive for cutaneous active vasodilatation in humans

The precise role of nitric oxide (NO) in cutaneous active vasodilatation in humans is unknown. We tested the hypothesis that NO is necessary to permit the action of an unknown vasodilator. Specifically, we investigated whether a low‐dose infusion of exogenous NO, in the form of sodium nitroprusside (SNP), would fully restore vasodilatation in an area of skin in which endogenous NO was inhibited during hyperthermia. This finding would suggest a ‘permissive’ role for NO in active vasodilatation. Eight subjects were instrumented with three microdialysis fibres in forearm skin. Sites were randomly assigned to (1) Site A: control site; (2) Site B: NO synthase (NOS) inhibition during established hyperthermia; or (3) Site C: NOS inhibition throughout the protocol. Red blood cell flux was measured using laser‐Doppler flowmetry (LDF) and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was normalized to maximal vasodilatation at each site. In Site B, NG‐nitro‐l‐arginine methyl ester (l‐NAME) infusion during hyperthermia reduced CVC by ∼32 % (65 ± 4 % CVCmaxvs. 45 ± 4 % CVCmax; P < 0.05). Vasodilatation was not restored to pre‐NOS inhibition values in this site following low‐dose SNP infusion (55 ± 4 % CVCmaxvs. 65 ± 4 % CVCmax; P < 0.05). CVC remained significantly lower than the control site with low‐dose SNP infusion in Site C (P < 0.05). The rise in CVC with low‐dose SNP (ΔCVC) was significantly greater in Site B and Site C during hyperthermia compared to normothermia (P < 0.05). No difference in ΔCVC was observed between hyperthermia and normothermia in the control site (Site A). Thus, NO does not act permissively in cutaneous active vasodilatation in humans but may directly mediate vasodilatation and enhance the effect of an unknown active vasodilator.

Adenosine receptor antagonist and augmented vasodilation during hypoxic exercise

We tested the hypothesis that adenosine contributes to augmented skeletal muscle vasodilation during hypoxic exercise. In separate protocols, subjects performed incremental rhythmic forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O2 saturation). In protocol 1 (n = 8), subjects received an intra-arterial administration of saline (control) and aminophylline (adenosine receptor antagonist). In protocol 2 (n = 10), subjects received intra-arterial phentolamine (alpha-adrenoceptor antagonist) and combined phentolamine and aminophylline administration. Forearm vascular conductance (FVC; in ml x min(-1).100 mmHg(-1)) was calculated from forearm blood flow (in ml/min) and blood pressure (in mmHg). In protocol 1, the change in FVC (DeltaFVC; change from normoxic baseline) during hypoxic exercise with saline was 172 +/- 29 and 314 +/- 34 ml x min(-1) x 100 mmHg(-1) (10% and 20%, respectively). Aminophylline administration did not affect DeltaFVC during hypoxic exercise at 10% (190 +/- 29 ml x min(-1)x100 mmHg(-1), P = 0.4) or 20% (287 +/- 48 ml x min(-1) x 100 mmHg(-1), P = 0.3). In protocol 2, DeltaFVC due to hypoxic exercise with phentolamine infusion was 313 +/- 30 and 453 +/- 41 ml x min(-1) x 100 mmHg(-1) (10% and 20% respectively). DeltaFVC was similar at 10% (352 +/- 39 ml min(-1) x 100 mmHg(-1), P = 0.8) and 20% (528 +/- 45 ml x min(-1) x 100 mmHg(-1), P = 0.2) hypoxic exercise with combined phentolamine and aminophylline. In contrast, DeltaFVC to exogenous adenosine was reduced by aminophylline administration in both protocols (P < 0.05 for both). These observations suggest that adenosine receptor activation is not obligatory for the augmented hyperemia during hypoxic exercise in humans.

Roles of nitric oxide synthase and cyclooxygenase in leg vasodilation and oxygen consumption during prolonged low-intensity exercise in untrained humans

The vasodilator signals regulating muscle blood flow during exercise are unclear. We tested the hypothesis that in young adults leg muscle vasodilation during steady-state exercise would be reduced independently by sequential pharmacological inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) with NG-nitro-L-arginine methyl ester (L-NAME) and ketorolac, respectively. We tested a second hypothesis that NOS and COX inhibition would increase leg oxygen consumption (VO2) based on the reported inhibition of mitochondrial respiration by nitric oxide. In 13 young adults, we measured heart rate (ECG), blood pressure (femoral venous and arterial catheters), blood gases, and venous oxygen saturation (indwelling femoral venous oximeter) during prolonged (25 min) steady-state dynamic knee extension exercise (60 kick/min, 19 W). Leg blood flow (LBF) was determined by Doppler ultrasound of the femoral artery. Whole body VO2 was measured, and leg VO2 was calculated from blood gases and LBF. Resting intra-arterial infusions of acetylcholine (ACh) and nitroprusside (NTP) tested inhibitor efficacy. Leg vascular conductance (LVC) to ACh was reduced up to 53±4% by L-NAME+ketorolac infusion, and the LVC responses to NTP were unaltered. Exercise increased LVC from 4±1 to 33.1±2 ml.min(-1).mmHg(-1) and tended to decrease after L-NAME infusion (31±2 ml.min(-1).mmHg(-1), P=0.09). With subsequent administration of ketorolac LVC decreased to 29.6±2 ml.min(-1).mmHg(-1) (P=0.02; n=9). While exercise continued, LVC returned to control values (33±2 ml.min(-1).mmHg(-1)) within 3 min, suggesting involvement of additional vasodilator mechanisms. In four additional subjects, LVC tended to decrease with L-NAME infusion alone (P=0.08) but did not demonstrate the transient recovery. Whole body and leg VO2 increased with exercise but were not altered by L-NAME or L-NAME+ketorolac. These data indicate a modest role for NOS- and COX-mediated vasodilation in the leg of exercising humans during prolonged steady-state exercise, which can be restored acutely. Furthermore, NOS and COX do not appear to influence muscle VO2 in untrained healthy young adults.

Nitric oxide-mediated vasodilation becomes independent of β-adrenergic receptor activation with increased intensity of hypoxic exercise

Hypoxic vasodilation in skeletal muscle at rest is known to include β-adrenergic receptor-stimulated nitric oxide (NO) release. We previously reported that the augmented skeletal muscle vasodilation during mild hypoxic forearm exercise includes β-adrenergic mechanisms. However, it is unclear whether a β-adrenergic receptor-stimulated NO component exists during hypoxic exercise. We hypothesized that NO-mediated vasodilation becomes independent of β-adrenergic receptor activation with increased exercise intensity during hypoxic exercise. Ten subjects (7 men, 3 women; 23 ± 1 yr) breathed hypoxic gas to titrate arterial O(2) saturation to 80% while remaining normocapnic. Subjects performed two consecutive bouts of incremental rhythmic forearm exercise (10% and 20% of maximum) with local administration (via a brachial artery catheter) of propranolol (β-adrenergic receptor inhibition) alone and with the combination of propranolol and nitric oxide synthase inhibition [N(G)-monomethyl-l-arginine (l-NMMA)] under normoxic and hypoxic conditions. Forearm blood flow (FBF, ml/min; Doppler ultrasound) and blood pressure [mean arterial pressure (MAP), mmHg; brachial artery catheter] were assessed, and forearm vascular conductance (FVC, ml·min(-1)·100 mmHg(-1)) was calculated (FBF/MAP). During propranolol alone, the rise in FVC (Δ from normoxic baseline) due to hypoxic exercise was 217 ± 29 and 415 ± 41 ml·min(-1)·100 mmHg(-1) (10% and 20% of maximum, respectively). Combined propranolol-l-NMMA infusion during hypoxic exercise attenuated ΔFVC at 20% (352 ± 44 ml·min(-1)·100 mmHg(-1); P < 0.001) but not at 10% (202 ± 28 ml·min(-1)·100 mmHg(-1); P = 0.08) of maximum compared with propranolol alone. These data, when integrated with earlier findings, demonstrate that NO contributes to the compensatory vasodilation during mild and moderate hypoxic exercise; a β-adrenergic receptor-stimulated NO component exists during low-intensity hypoxic exercise. However, the source of the NO becomes less dependent on β-adrenergic mechanisms as exercise intensity increases.

Autonomic Cardiovascular Control During a Novel Pharmacologic Alternative to Ganglionic Blockade

The purpose of this study was to compare ganglionic blockade with trimethaphan (TMP) and an alternative drug strategy using combined muscarinic antagonist (glycopyrrolate, GLY) and α‐2 agonist (dexmedetomidine, DEX). Protocol 1: incremental phenylephrine was administered during control and combined GLY‐DEX, or control and TMP on two control combined GLY and DEX or TMP infusion on two randomized days. Protocol 2: muscle sympathetic nerve activity (MSNA) and the baroreflex MSNA relationship was determined before and after GLY–DEX. Blood pressure was higher with GLY–DEX (99±3 mm Hg) and lower with TMP (78±3 mm Hg) relative to control (GLY–DEX: 90±2 mm Hg; TMP: 91±2 mm Hg;P<0.05). Incremental phenylephrine increased pressure during GLY–DEX (P<0.01 vs control) and TMP (P<0.01 vs control) to a similar degree. Both GLY–DEX and TMP infusion inhibited norepinephrine release (P<0.01 vs control). GLY–DEX inhibited baseline MSNA (P<0.05) and baroreflex changes in MSNA (P<0.01). We conclude that the GLY–DEX alternative drug strategy can be used as a reasonable alternative to pharmacologic ganglionic blockade to examine autonomic cardiovascular control.

Bring on the heat: transient receptor potential vanilloid type‐1 (TRPV‐1) channels as a sensory link for local thermal hyperaemia

Beyond the integumentary system role of human skin, the vascular control of the largest continuous organ in the human body contributes to the defence from temperature extremes. During exercise and/or exposure to high environmental heat, the skin blood flow defence to elevated whole body temperature includes a sympathetic active vasodilator (non-acral skin) pathway which involves the complex integration of neural and local mechanisms. In addition to reflex control of the cutaneous circulation during whole body thermal stress, non-painful locally applied heat evokes a substantial elevation in skin blood flow to near maximal values (Kellogg et al. 1999; Minson et al. 2001). The pattern of this robust vasodilatation can be predictable and is characterized by a distinct initial peak to a nadir before stabilizing at a prolonged plateau. The predictable nature of this response has led to an increased clinical application for describing generalized microvascular function. An altered skin blood flow pattern, or altered contribution of the substances involved, is observed with natural ageing and numerous diseases directly or indirectly linked to microvascular pathologies (Holowatz et al. 2008). The popularization of local skin heating protocols as a clinical tool necessitates a better understanding of the pathways involved. Collectively, research over the last 10 years has identified mechanisms related to efferent responses in human skin with local thermal stimuli. Many experiments dissecting these mechanisms have taken advantage of intradermal microdialysis techniques in combination with laser Doppler flowmetry. These methods allow pharmacological manipulation in a small area of skin (microdialysis), where the subsequent blood flow parameters can be non-invasively monitored (laser Doppler flowmetry). Using these techniques, the mechanisms contributing to this predictable blood flow pattern during local skin heating have been identified to some degree. The initial peak and nadir are dependent upon local axon reflexes, as application of EMLA cream on the skin surface (for non-specific cutaneous nerve block) substantially reduces this steep initial rise and peak in skin blood flow and uncouples the initial peak and nadir from the sustained plateau (Minson et al. 2001). The sustained plateau is dependent upon a robust NO-dependent vasodilatation (Kellogg et al. 1999; Minson et al. 2001). However, a NO-independent elevation in skin blood flow remains during NO synthase inhibition. The integration and interaction of signals from local axon reflexes and signals from vascular sources (i.e. NO) is complex. For example, NO modestly contributes to the intitial peak and nadir but only in the absence of cutaneous nerve blockade with EMLA cream (Minson et al. 2001). With these previously described mechanisms in mind, the investigation of Wong & Fieger (2010) in this issue of The Journal of Physiology has taken up the challenge to identify a link between afferent sensory nerves, responding to local heat application, and the resultant hyperaemia in human skin. To that end, they have demonstrated the activation of a transient receptor potential vanilliod (TRPV-1) channel to be compulsory for the full expression of the thermal hyperaemia to local heating. The TRPV-1 channels are located primarily on afferent sensory nerves in human skin and can be activated by a host of stimuli, including direct heat (Caterina, 2007). The authors’ clever, yet simple approach to the question incorporated pharmacological inhibition of TRPV-1 channels with capsazepine (specific TRPV-1 inhibition) via intradermal microdialysis. In contrast to previous work with EMLA cream, which would render all cutaneous nerves unresponsive, capsazepine appears to specifically inhibit TRVP-1 channels on cutaneous afferent sensory nerves. Using this approach, the authors meticulously investigated the direct effect of TRPV-1 channel inhibition on the resultant local cutaneous hypereamia and the interaction between TRPV-1 channels and NO production. The results clearly suggest a role for TRPV-1 channels, linking the thermal stimulus on the skin surface to the ensuing skin blood flow response. More specifically, TRPV-1 channel inhibition substantially reduced the axon reflex mediated portion of this blood flow pattern, i.e. the initial peak and nadir, while the plateau phase of the response was only modestly affected by TRPV-1 channel inhibition. These data extend what is currently known about the cutaneous blood flow response to local heating. The TRPV-1 channels may be directly activated by heat, thereby depolarizing afferent sensory nerves to initiate the axon reflex portion of the local thermal hyperaemia (Wong & Fieger, 2010). There was also an interesting interaction found between the TRPV-1 channel activation and the role for NO in this response. Worth noting, both the investigation of Wong & Fieger (2010) and the work of Minson et al. (2001) failed to completely block axon reflex mediated skin blood flow responses with capsazepine and EMLA cream, respectively. The question therefore remains, was there an incomplete pharmacological blockade or are there additional mechanisms which contribute to the axon reflex portion of the skin blood flow response? Taken in a wider context, the study by Wong and Fieger went beyond a continued reductionist approach to further dissect the mechanisms for the vascular responses to thermal stimulus in human skin. Their data begin to integrate the afferent sensory mechanisms with the efferent vascular control mechanisms for a more complete description of the skins defence against external heat application. For this, the authors are to be commended.