Pressor Reflex Research Articles

Effects of sex and ageing on the human respiratory muscle metaboreflex.

Intense inspiratory muscle work evokes a sympathetically mediated pressor reflex, termed the respiratory muscle metaboreflex, in which young females demonstrate an attenuated response relative to males. However, the effects of ageing and female sex hormones on the respiratory muscle metaboreflex are unclear. We tested the hypothesis that the pressor response to inspiratory work would be similar between older males and females, and higher relative to their younger counterparts. Healthy, normotensive young (26±3years) males (YM; n=10) and females (YF; n=10), as well as older (64±5years) males (OM; n=10) and females (OF; n=10), performed inspiratory pressure threshold loading (PTL) to task failure. Older adults had a greater mean arterial pressure (MAP) response to PTL than young (P<0.001). YF had a lower MAP compared to YM (+10±6 vs. +19±15mmHg, P=0.026); however, there was no difference observed between OF and OM (+26±11 vs. +27±11mmHg, P=0.162). Older adults had a lower heart rate response to PTL than young (P=0.002). There was no effect of sex between young females and males (+19±9 and +27±11bpm, P=0.186) or older females and males (+17±7 and +20±7bpm, P=0.753). We conclude the respiratory muscle metaboreflex response is heightened in older adults, and the sex effect between older males and post-menopause females is absent, suggesting an effect of circulating sex hormones. KEY POINTS: The arterial blood pressure response to the respiratory muscle metaboreflex is greater in older males and females. Compared to sex-matched young individuals, there is no sex differences in the blood pressure response between older males and post-menopause females. Our results suggest the differences between males and females in the cardiovascular response to high levels of inspiratory muscle work is abolished with reduced circulating female sex hormones.

Bradykinin 2 receptors contribute to the exaggerated exercise pressor reflex in a rat model of simulated peripheral artery disease.

We investigated the role played by bradykinin 2 (B2) receptors in the exaggerated exercise pressor reflex in rats with a femoral artery ligated for 72 h to induce simulated peripheral artery disease (PAD). We hypothesized that in decerebrate, unanesthetized rats with a ligated femoral artery, hindlimb arterial injection of HOE-140 (100 ng, B2 receptor antagonist) would reduce the pressor response to 30 s of electrically induced 1 Hz hindlimb skeletal muscle contraction, and 30 s of 1 Hz hindlimb skeletal muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production). We hypothesized no effect of HOE-140 in sham-operated "freely perfused" rats. In both freely perfused (n = 4) and "ligated" (n = 4) rats, we first confirmed efficacious B2 receptor blockade by demonstrating that HOE-140 injection significantly reduced (P < 0.05) the peak increase in mean arterial pressure (peak ΔMAP) in response to hindlimb arterial injection of bradykinin. In subsequent experiments, we found that HOE-140 reduced the peak ΔMAP response to muscle contraction in ligated (n = 14; control: 23 ± 2; HOE-140: 17 ± 2 mmHg; P = 0.03) but not freely perfused rats (n = 7; control: 17 ± 3; HOE-140: 18 ± 4 mmHg; P = 0.65). Furthermore, HOE-140 had no effect on the peak ΔMAP response to stretch in ligated rats (n = 14; control: 37 ± 4; HOE-140: 32 ± 5 mmHg; P = 0.13) but reduced the integrated area under the blood pressure signal over the final ∼20 s of the maneuver. The data suggest that B2 receptors contribute to the exaggerated exercise pressor reflex in rats with simulated PAD, and that contribution includes a modest role in the chronic sensitization of the mechanically activated channels/afferents that underlie mechanoreflex activation.

Renal artery denervation: unsolved questions about mechanisms of changes of systemic hemodynamics

In the review we discussed the clinical and physiological aspects of the use of renal artery denervation as an interventional method of treatment of refractory essential arterial hypertension. The application of radiofrequency denervation may be considered as an additional treatment to drug therapy in patients with arterial hypertension. At the same time, the main emphasis in the performed experimental and clinical studies was made on the assessment of changes in blood pressure, cardiac output and the calculated index of total peripheral resistance. However, there is no satisfactory data in the literature about changes of the venous return, as well as superior and inferior venae cavae flows. To assess the effectiveness of renal artery denervation on arterial and venous vessels resistance shifts, it is necessary to carry out experimental studies with perfusion of isolated organs in the conditions of modeling arterial hypertension in animals. The question of the extent to which denervation of the renal arteries leads to shifts in the reactions of organ venous vessels in response to pressor reflex stimuli also requires further studies on experimental models. There are practically no data in the literature on the degree of influence of renal artery denervation on orthostatic tolerance. To understand the mechanisms of changes in systemic hemodynamics in response to denervation of the renal arteries, it is also necessary to perform further research on the mechanisms of shifts in the capacitive and resistive functions of the pulmonary circulation with an assessment of pulmonary microcirculation.

Sympathetic Nerve Activity and Blood Pressure Response to Exercise in Peripheral Artery Disease: From Molecular Mechanisms, Human Studies, to Intervention Strategy Development.

Sympathetic nerve activity (SNA) regulates the contraction of vascular smooth muscle and leads to a change in arterial blood pressure (BP). It was observed that SNA, vascular contractility, and BP are heightened in patients with peripheral artery disease (PAD) during exercise. The exercise pressor reflex (EPR), a neural mechanism responsible for BP response to activation of muscle afferent nerve, is a determinant of the exaggerated exercise-induced BP rise in PAD. Based on recent results obtained from a series of studies in PAD patients and a rat model of PAD, this review will shed light on SNA-driven BP response and the underlying mechanisms by which receptors and molecular mediators in muscle afferent nerves mediate the abnormalities in autonomic activities of PAD. Intervention strategies, particularly non-pharmacological strategies, improving the deleterious exercise-induced SNA and BP in PAD, and enhancing tolerance and performance during exercise will also be discussed.

The Respiratory Muscle Metaboreflex: Effects Of Sex And Aging

High levels of respiratory muscle work evoke a sympathetically mediated pressor reflex (e.g. metaboreflex), and young females demonstrate an attenuated heart rate and blood pressure response relative to males. It is unclear what the inter-relationships are between the effects of healthy aging and female sex hormones on the respiratory muscle metaboreflex. PURPOSE: We tested the hypothesis that the pressor response to inspiratory work would be similar between older men and women, and higher relative to their younger counterparts. METHODS: Healthy, normotensive young (26 ± 3 y) males (YM; n = 10) and females (YF; n = 9) and aged (63 ± 6 y) males (OM; n = 5) and females (OF; n = 10, post-menopausal) participated. YF were tested in the mid-luteal phase of the menstrual cycle. Participants performed inspiratory pressure threshold loading (PTL; 60% maximal inspiratory mouth pressure, breathing frequency = 15 bpm, 0.70 inspiratory duty cycle) to task failure. Mean arterial pressure (MAP) and heart rate (HR) were recorded continuously. RESULTS:Time to task failure was different between groups (YF = 19 ± 8. YM = 18 ± 9, OF = 13 ± 6, OM = 8 ± 5 min, p = 0.036). The absolute pressure time product, per minute was also different between groups (p = 0.005); of which, OM generated significantly greater pressures than YW (p = 0.018). There was a significant age (p = 0.002) and sex (p = 0.030) effect in the MAP response to PTL. Peak MAP between OF (+25 ± 10 mmHg) and OM (+17 ± 4 mmHg) were similar (p = 0.999). Peak MAP in OF was greater than YF (+8 ± 6 mmHg, p = 0.010) and was similar to YM (+19 ± 15 mmHg, p = 0.475). The peak MAP response was greater in YM compared to YW (p = 0.009). There was a significant sex, time interaction in the HR response to PTL (p = 0.007). YM showed the greatest peak HR response to PTL (+27 ± 11 bpm) though not significantly different to YF (+15 ± 12 bpm), OF (+17 ± 7 bpm), or OM (+18 ± 6 bpm). CONCLUSIONS:Healthy aging is associated with a heightened respiratory muscle metaboreflex compared to young men and women. The cardiovascular response to the respiratory muscle metaboreflex is similar between older males and post-menopausal females.Support: NSERC

Brain Insulin Receptor Antagonism Augments The Blood Pressure Response To Activation Of The Exercise Pressor Reflex

Type 2 diabetes mellitus (T2DM) results in abnormal control of blood pressure (BP). However, the underlying mechanisms remain to be elucidated. The transport of insulin into the central nervous system is decreased in T2DM. The resultant central hypoinsulinemia reduces neuronal excitability. The nucleus tractus solitarius (NTS) is a key central integration site for incoming signals from peripheral sensory neurons including input from working skeletal muscle and arterial baroreceptors. Working skeletal muscle activates primary sensory fibers and evokes the exercise pressor reflex (EPR) to reflexively increase BP, a response modulated by the baroreflex. Central hypoinsulinemia in T2DM may influence how neurons in the NTS process this sensory information during exercise affecting BP responsiveness. PURPOSE: To determine whether acute antagonism of insulin receptors in the NTS alters EPR function in baro-intact and barodenervated rats. METHODS: L4 and L5 ventral roots were electrically stimulated in healthy decerebrated male Sprague-Dawley rats to evoke hindlimb muscle contraction and EPR activation in intact and sino-aortic barodenervated rats. Mean arterial pressure (MAP) responses to activation of the EPR were assessed before and 30 min following NTS microinjections of the insulin receptor antagonist, GSK1838705 (100uM), or vehicle, dimethyl sulfoxide (DMSO). RESULTS: In baro-intact rats, contraction-induced changes in MAP were significantly greater following GSK1838705 (Pre, 16 ± 10 mmHg vs. Post, 23 + 13 mmHg, n = 11, p < 0.01) but not DMSO (Pre, 15 ± 8 mmHg vs. Post, 15 + 8 mmHg, n = 9). In barodenervated rats, neither GSK1838705 (Pre, 27 ± 16 mmHg vs. Post, 28 ± 21 mmHg, n = 8) nor DMSO (Pre, 34 ± 17 mmHg vs. Post, 30 ± 27 mmHg, n = 9) affected the MAP response to contraction. CONCLUSIONS: The findings demonstrate that central insulin receptor antagonism augments the BP response to EPR activation, which is abolished by barodenervation. The findings also suggest that insulin in the NTS maintains the capacity to buffer EPR-mediated increases in BP via baroreflex-mediated mechanisms. Alterations in this buffering capacity due to the central hypoinsulinemia in T2DM could account, in part, for the exaggerated pressor response to exercise in this disease. Supported by NIH-NHLBI, Lawson & Rogers Lacy Research Fund.

Adjusting For Exercise Intensity Attenuates Sex Differences In Blood Pressure During Exercise In Healthy Adults

PURPOSE: The exercise pressor reflex, composed of the muscle mechano- and metaboreflex, increases blood pressure (BP). Compared to males, healthy female adults typically exhibit blunted BP responses to exercise. However, recent work suggests that sex differences in BP during isometric handgrip (HG) exercise and post-exercise ischemia (PEI; metaboreflex isolation) are attenuated after adjusting for differences in maximal voluntary contraction (MVC). Therefore, the purpose of this study was to determine whether sex differences in BP responses during HG and PEI would be attenuated after adjustment for submaximal HG squeezing force. METHODS: We analyzed data from 95 participants including 34 females (age: 24±4 years, BMI: 24±4 kg/m2, screening BP: 106±13/64±9 mmHg, Mean ± SD) and 61 males (age: 25±5 years, BMI: 2±3 kg/m2, screening BP: 113±11/66±9 mmHg). All females were tested during days 1-5 of their menstrual cycle. Maximal HG force for each participant was defined as the average of three MVCs. Following a 10-minute baseline period, participants performed static static HG exercise at 40% of their average MVC for two minutes followed by three minutes of PEI via brachial occlusion. We obtained beat-to-beat BP readings via finger photoplethysmography (Finometer). Statistical analyses included t-tests, 2-way ANOVAs (sex x time), and ANCOVAs (average HG force as a covariate). RESULTS: Females exhibited a lower absolute 40% HG force than males (105±30 N v. 169±45 N, p<0.001). There was a significant sex x time interaction for ∆ systolic BP during HG (e.g., minute 2, females: 20±12 mmHg v. 28±18 mmHg in males, p=0.014), but not ∆ diastolic (p=0.106) or ∆ mean BP (p=0.058). After adjusting for absolute HG force, the sex difference for peak ∆ systolic BP during HG was attenuated (p=0.072). During PEI there were sex x time interactions for ∆ systolic, ∆ diastolic, and ∆ mean BP (p<0.05 for all) and adjustment for HG force did not attenuate these differences. Additionally, in a small strength-matched cohort (N=48, 29 females, female HG force mean = 107±23 N, male HG force mean = 118 ± 24 N, p=0.092) there was not a significant sex difference for peak ∆ systolic BP during HG (p=0.667) or PEI (p=0.828). CONCLUSIONS: Our data indicate that the sex difference in BP reactivity is attenuated after adjusting for absolute HG force during exercise.

NaV1.9 current in muscle afferent neurons is enhanced by substances released during muscle activity.

Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (NaV) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R NaV currents. The rise time of this enhancement averaged ∼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support NaV1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced NaV current, but the sum of these enhancements was <1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance NaV1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of NaV1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR.NEW & NOTEWORTHY G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that NaV1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of NaV1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

Abstract P3134: Mechanosensitive Piezo2 Channel Mediates The Exaggerated Exercise Pressor Reflex In A Mouse Model Of Hind Limb Ischemia.

The exercise pressor reflex (EPR) is a reflexive mechanism in which blood pressure and heart rate increase in response to skeletal muscle contraction. These reflexive increases are caused by both mechanical and metabolic stimulation of group III and IV afferent neurons innervating in contracting skeletal muscle. Patients with peripheral artery disease (PAD) have an exaggerated EPR which contributes to exercise intolerance and morbidity and mortality. Recently, we developed a mouse model of EPR, and have validated that the mouse model is similar to our rat model. With this mouse model of EPR, we studied the EPR following femoral artery ligation (FAL), briefly the right hind limb common femoral artery was ligated and transected between the proximal end near the groin and the distal end near the bifurcation of the popliteal artery and the saphenous artery. The same procedure was done without femoral artery ligation and transection for sham control. We observed that the mean arterial pressure (MAP) responses to ventral root stimulation (which excite metabo- and mechano-sensitive afferent neurons) and passive stretch (mechanosensitive stimulation only) were exaggerated 72 hours following FA ligation when compared to sham treated controls. The exaggerated EPR was significantly reduced by intra-arterial administration of Gadolinium (a mechanical sensitive group III afferent neurons inhibitor), GsMTx-4 (a selective Piezo2 inhibitor), but not by SB-705498 (a TRPv1 receptor inhibitor). Additionally, selective stimulation of metabolically sensitive afferent neurons by intra-arterial capsaicin injection (an activator of metabolically sensitive afferent neurons) induced equal, dose-related increases in MAP in both FA ligation and control mice. Finally, immunofluorescence staining visualized that the mechano-sensitive channel Piezo2 is localized in most large diameter neuron cells in mouse lumbar DRG. First, these results indicate that metabolically sensitive afferent neurons are not affected by FAL. Second, we observed that the exaggerated EPR in a murine model of PAD is mediated by mechanically sensitive group III neurons, which is due, in part, to Piezo2 receptor activation. Future studies will be aimed at interrogation of the mechanosensitive mechanisms that mediate this abnormal response to exercise in a model of PAD. The goal of these studies will be to determine molecular targets through which the exaggerated EPR can be normalized to allow for the prescription of exercise in PAD patients.

Dapagliflozin Attenuates Sympathetic and Pressor Responses to Stress in Young Prehypertensive Spontaneously Hypertensive Rats.

SGLT2i (sodium-glucose cotransporter 2 inhibitor), a class of anti-diabetic medications, is shown to reduce blood pressure (BP) in hypertensive patients with type 2 diabetes. Mechanisms underlying this action are unknown but SGLT2i-induced sympathoinhibition is thought to play a role. Whether SGLT2i reduces BP and sympathetic nerve activity (SNA) in a nondiabetic prehypertension model is unknown. Accordingly, we assessed changes in conscious BP using radiotelemetry and alterations in mean arterial pressure and renal SNA during simulated exercise in nondiabetic spontaneously hypertensive rats during chronic administration of a diet containing dapagliflozin (0.5 mg/kg per day) versus a control diet. We found that dapagliflozin had no effect on fasting blood glucose, insulin, or hemoglobin A1C levels. However, dapagliflozin reduced BP in young (8-week old) spontaneously hypertensive rats as well as attenuated the age-related rise in BP in adult spontaneously hypertensive rat up to 17-weeks of age. The rises in mean arterial pressure and renal SNA during simulated exercise (exercise pressor reflex activation by hindlimb muscle contraction) were significantly reduced after 4 weeks of dapagliflozin (Δmean arterial pressure: 10±7 versus 25±14 mm Hg, Δrenal SNA: 31±17% versus 68±39%, P<0.05). Similarly, rises in mean arterial pressure and renal SNA during mechanoreflex stimulation by passive hindlimb stretching were also attenuated by dapagliflozin. Heart weight was significantly decreased in dapagliflozin compared with the control group. These data demonstrate a novel role for SGLT2i in reducing resting BP as well as the activity of skeletal muscle reflexes, independent of glycemic control. Our study may have important clinical implications for preventing hypertension and hypertensive heart disease in young prehypertensive individuals.

Head hemodynamics and systemic responses during auditory stimulation.

The present study aims to analyze the systemic response to auditory stimulation by means of hemodynamic (cephalic and peripheral) and autonomic responses in a broad range of auditory intensities (70.9, 77.9, 84.5, 89.5, 94.5 dBA). This approach could help to understand the possible influence of the autonomic nervous system on the cephalic blood flow. Twenty‐five subjects were exposed to auditory stimulation while electrodermal activity (EDA), photoplethysmography (PPG), electrocardiogram, and functional near‐infrared spectroscopy signals were recorded. Seven trials with 20 individual tones, each for the five intensities, were presented. The results showed a differentiated response to the higher intensity (94.5 dBA) with a decrease in some peripheral signals such as the heart rate (HR), the pulse signal, the pulse transit time (PTT), an increase of the LFnu power in PPG, and at the head level a decrease in oxygenated and total hemoglobin concentration. After the regression of the visual channel activity from the auditory channels, a decrease in deoxyhemoglobin in the auditory cortex was obtained, indicating a likely active response at the highest intensity. Nevertheless, other measures, such as EDA (Phasic and Tonic), and heart rate variability (Frequency and time domain) showed no significant differences between intensities. Altogether, these results suggest a systemic and complex response to high‐intensity auditory stimuli. The results obtained in the decrease of the PTT and the increase in LFnu power of PPG suggest a possible vasoconstriction reflex by a sympathetic control of vascular tone, which could be related to the decrease in blood oxygenation at the head level.

A comparison of magnesium sulfate plus morphine with morphine alone for acute pain reduction in long-bone fractures; a double blind randomized clinical trial

Introduction: Acute pain is a common symptom in patients with bone fractures referring to the emergency department (ED). Objectives: The present study aimed at evaluating the efficacy of intravenous (IV) magnesium sulfate in controlling acute pain of long-bone fractures in EDs. Patients and Methods: The present double-blind, randomized clinical trial was conducted on patients with isolated limb fractures referred to Golestan hospital of Ahvaz from 2019 to 2020. Patients in the intervention group were injected with 0.1 mg/kg morphine plus 30 mg/kg magnesium sulfate and 0.1 mg/kg morphine alone in the morphine group. The patient’s pain scores were measured at 0, 15, 30, 45 and 60 minutes after injection. The patients’ condition was monitored every 10 minutes, including respiratory and pulse rate, blood pressure, and knee reflex. Results: The present study was conducted on 64 patients with long-bone fractures allocated to two groups of IV morphine plus magnesium sulfate and IV morphine alone (each of 32). The hemodynamic factors and mean body temperature did not significantly change after intervention and had a similar level in both groups. Before the intervention, the mean score of pain was measured in the morphine and magnesium sulfate plus morphine groups using a visual analogue scale. Results which were 8.87±0.83 and 8.93±0.94, respectively, showing no significant differences (P=0.69). After 60 minutes, the pain score reduced considerably in both groups. Conclusion: In the study, magnesium sulfate plus morphine could better reduce pain than morphine alone without influencing the respiratory rate. Trial Registration: The trial protocol was approved by the Iranian Registry of Clinical Trials (IRCT20190617043913N1; https://en.irct.ir/trial/40370, ethical code# IR.AJUMS.REC.1398.143).

The exercise pressor reflex: An update.

The exercise pressor reflex is a feedback mechanism engaged upon stimulation of mechano- and metabosensitive skeletal muscle afferents. Activation of these afferents elicits a reflex increase in heart rate, blood pressure, and ventilation in an intensity-dependent manner. Consequently, the exercise pressor reflex has been postulated to be one of the principal mediators of the cardiorespiratory responses to exercise. In this updated review, we will discuss classical and recent advancements in our understating of the exercise pressor reflex function in both human and animal models. Particular attention will be paid to the afferent mechanisms and pathways involved during its activation, its effects on different target organs, its potential role in the abnormal cardiovascular response to exercise in diseased states, and the impact of age and biological sex on these responses. Finally, we will highlight some unanswered questions in the literature that may inspire future investigations in the field.

Gene and protein expression of dorsal root ganglion sensory receptors in normotensive and hypertensive male rats.

The exercise pressor reflex (EPR), a neurocirculatory control mechanism, is exaggerated in hypertensive humans and rats. Disease-related abnormalities within the afferent arm of the reflex loop, including mechano- and metabosensitive receptors located at the terminal end of group III/IV muscle afferents, may contribute to the dysfunctional EPR in hypertension. Using control (WKY) and spontaneous hypertensive (SHR) rats, we examined dorsal root ganglion (DRG) gene and protein expression of molecular receptors recognized as significant determinants of the EPR. Twelve lumbar DRGs (6 left, 6 right) were harvested from each of 10 WKY [arterial blood pressure (MAP): 96 ± 9 mmHg] and 10 SHR (MAP: 144 ± 9 mmHg). DRGs from the left side were used for protein expression (Western blotting; normalized to GAPDH), whereas right-side DRGs (i.e., parallel structure) were used to determine mRNA levels (RNA-sequencing, normalized to TPM). Analyses focused on metabosensitive (ASIC3, Bradykinin receptor B2, EP4, P2X3, TRPv1) and mechanosensitive (Piezo1/2) receptors. Although Piezo1 was similar in both groups (P = 0.75), protein expression for all other receptors was significantly higher in SHR compared with WKY. With the exception of a greater Bradykinin-receptor B2 in SHR (P < 0.05), mRNA expression of all other receptors was not different between groups (P > 0.18). The higher protein content of these sensory receptors in SHR indirectly supports the previously proposed hypothesis that the exaggerated EPR in hypertension is, in part, due to disease-related abnormalities within the afferent arm of the reflex loop. The upregulated receptor content, combined with normal mRNA levels, insinuates that posttranscriptional regulation of sensory receptor protein expression might be impaired in hypertension.

The Effect of Amiloride (10mg) on the Blood Pressure Response to Ischemic Exercise: Preliminary Observations

BackgroundDuring exercise, blood pressure increases via augmented central command and stimulation of mechano‐ and metabosensitive muscle afferents (i.e., the exercise pressor reflex). Recent evidence has reported a strong role of acid sensing ion channels (ASIC’s) in mediating this reflex, as well as exercise induced muscle pain. Therefore, it is logical to suspect that inhibiting ASIC’s would mitigate the exaggerated blood pressure responses and exercise related muscle pain observed in individuals with peripheral artery disease (PAD).Purpose. We tested the hypothesis that a single 10mg dose of amiloride (a K+‐sparing diuretic known to inhibit ASIC’s) would attenuate the pressor response to blood flow restricted (BFR) plantar flexion exercise in young healthy adults.MethodsSix healthy subjects (5 male, 26 ± 2 y/o, 180.8 ± 12.1 cm, 79.7 ± 12.9 Kg) completed three separate visits. Each visit included two bouts of isometric plantar flexion, both performed until failure at 30% of the predetermined maximal voluntary isometric contraction. During one bout, blood flow to the limb was restricted via a pressurized cuff placed around the proximal portion of the lower leg (BFR); the other bout was performed under free flow conditions. In the first visit, subjects performed these two exercise bouts (in random order) under normal, non‐treated conditions. In the second and third visits, subjects performed these exercise bouts (same order as in visit 1) ~3 hrs after oral ingestion of 10mg of amiloride, or ingestion of a placebo (random order). Blood pressure and heart rate were continuously recorded throughout exercise, and time to fatigue and muscle pain (on a 12cm visual analog scale) was recorded at the end of each exercise bout. Only data from the placebo and amiloride conditions are presented here.ResultsThe addition of BFR reduced total exercise time by ~40.8% (t=2.99, p=0.03), but had no significant effect on peak muscle pain (p&gt;0.10). Due to the preliminary nature of these data, no significant main effects of treatment (amiloride vs. placebo) were observed for free flow or BFR exercise (all p&gt;0.05). However, we did observe a net decrease in the average heart rate response (+10.7 ± 3.0 bpm vs. +11.3 ± 3.7 bpm, ηp2=0.539, p&gt;0.47), the total blood pressure response (5477.5 ± 373.7 mmHg*sec vs. 5841.3 ± 518.9 mmHg*sec, ηp2=0.250, p&gt;0.30), the total time‐tension index (595.9 ± 406.5 Kg*sec vs. 691.6 ± 394.2 Kg*sec, ηp2=0.211, p&gt;0.30), and the blood pressure to time‐tension index relationship (9.09 ± 4.40 mmHg/Kg vs. 11.38 ± 6.30 mmHg/Kg, ηp2=0.08, p&gt;0.50) in the amiloride condition compared to the placebo condition during BFR exercise.ConclusionsWhile these preliminary findings do not indicate significant effects of treatment, these observations serve as strong pilot data to support the continued investigation of amiloride as a method of attenuating the pressor response to ischemic exercise.

Dietary nitrate supplementation attenuates the mechanoflex in older women with early menopause: A pilot study

Women who reached early menopause (EM, 40‐44 yrs) have a greater risk for cardiovascular disease than women with normal menopause (NM, 50‐54 yrs), which may be attributed to a longer duration of estrogen loss and a greater reduction in nitric oxide bioavailability in both the peripherals and center. Previous studies have shown that dietary nitrate supplementation improves cardiovascular function in older women, but how autonomic neural control changes in daily living, such as during exercise, has been involved is unclear. We examined the hypothesis that 2wks continuous use of dietary nitrate supplements would attenuate the exercise pressor reflex in older women with EM.Six EM (65±4 [SD] yrs) and 8 NM (64±2 yrs) women participated in this randomized, placebo‐controlled, double‐blind, cross‐over pilot study. All subjects underwent 2 wks of beetroot juice (BRJ; 140 ml containing ~800 mg nitrate per day) or placebo (140 ml nitrate‐depleted BRJ, daily) intervention with ≥14 days of wash‐out. Before and after each intervention period, subjects performed SHG using the dominant hand at 40% of maximum voluntary contraction (MVC) until fatigue, followed by 2 min of PECA with an upper arm cuff inflated to 250 mmHg. Heart rate (HR; ECG), blood pressure (BP; finger photoplethysmography), muscle sympathetic nerve activity (MSNA; microneurography), and respiration (capnography) were continuously measured. In addition, 24‐h ambulatory BP standard deviation (SD) was monitored before and after each intervention (an indication of BP variability).There were no differences in baseline HR, BP and MSNA, MVC, and the time to fatigue between the EM and NM groups before and after intervention (all p&gt;0.05). At the first 30 s of SHG, the EM group showed significant decreases in MSNA burst frequency (DBF; ‐4.5±6.5 in EM vs. 2.5±6.2 bursts/min in NM, p=0.065) and burst incidence (DBI; ‐10.6±12.6 vs. 2.6±8.4 bursts/100 heartbeats, p=0.037) after BRJ intervention compared with the NM group (Figure1A and B). However, there were no differences between groups at fatiguing exercise and during PECA after the intervention. The interventional changes in 24‐h diastolic BP SD had a positive correlation with ΔMSNA BF (r=0.55, p=0.053) and BI (r=0.58, p=0.036) at the first 30 s of SHG in all subjects and EM individuals were in the left‐lower area (Figure2 A and B).These results suggest that dietary nitrate supplementation may attenuate and mechanoreflex, but not the metaboreflex, in older women with EM during static hand grip, which has a relation with BP variability during ambulation. Certainly, these preliminary observations need to be verified in more older women in future studies.

Pro‐inflammatory Cytokine IL‐1β Contributes to the Exaggerated Exercise Pressor Reflex in UC, Davis‐Type 2 Diabetes Mellitus Rats

Studies have shown that pressor responses to muscle contraction and tendon stretch are exaggerated in type 2 diabetes mellitus (T2DM). Low‐grade inflammation, which contributes to the underlying pathology of T2DM, involves an increase in pro‐inflammatory cytokines. These cytokines are known to play a role in exaggerating the pressor response during muscle contraction and tendon stretch. Although a previous study found that the pro‐inflammatory cytokine, interleukin‐1 beta (IL‐1β) is increased in the serum of individuals with T2DM, it is not known whether changes in IL‐1β in the presence of low‐grade inflammation play a role in exaggerating the exercise pressor reflex in T2DM. Therefore, the purpose of this study was to determine the effects of IL‐1β on the pressor responses evoked by skeletal muscle contraction and tendon stretch in T2DM rats. We hypothesized that IL‐1β plays a role in exaggerating the pressor response to muscle contraction and tendon stretch in T2DM rats.MethodsThe exercise pressor reflex was evoked by statically and intermittently contracting the hindlimb for 30 s, and the mechanoreflex was evoked by passively stretching the Achilles tendon for 30 s. We first evoked the exercise pressor reflex and mechanoreflex before and after activating IL‐1 receptors by injecting IL‐1β (5ng/uL) into the hindlimb circulation of working skeletal muscles in healthy unanesthetized, decerebrated male Sprague Dawley rats. We then evoked the exercise pressor reflex and mechanoreflex before and after blocking IL‐1 receptors using Kineret (1ug/100ul), an IL‐1 receptor antagonist, in fasted, unanesthetized, decerebrated male UCD‐T2DM rats. Peak changes in mean arterial pressure and heart rate were measured and compared before and after injecting IL‐1β or Kineret.ResultsIn healthy rats, activating IL‐1 receptors by injecting IL‐1β significantly increased the peak pressor response to static contraction (before: 12 ± 1, after: 24 ± 2 mmHg, n=8, p&lt;0.05) and intermittent contraction (before: 9 ± 1, after: 17 ± 2 mmHg, n=8, p&lt;0.05), but not stretch (before: 12 ± 2, after: 13 ± 2, n=9, p=0.23), although the cardioaccelerator response to intermittent contraction was significantly lower after IL‐1β injection (before: 18 ± 3, after: 14 ± 2 bpm, n=8, p&lt;0.05). Furthermore, in UCD‐T2DM rats, blocking IL‐1 receptors significantly attenuated the pressor and cardioaccelerator responses to static contraction (pressor: before: 29 ± 4, after: 13 ± 3 mmHg; cardioaccelerator: before: 36 ± 8, after: 14 ± 2 bpm; n=5, p&lt;0.05), intermittent contraction (pressor: before: 23 ± 5, after: 9 ± 2 mmHg; cardioaccelerator: before: 43 ± 11, after: 21 ± 6 bpm n=4, p&lt;0.05), and stretch (pressor: before: 22 ± 9, after: 13 ± 6 mmHg; cardioaccelerator: before: 34 ± 8, after: 14 ± 4 bpm n=4, p&lt;0.05). The developed tension was similar for all comparisons.ConclusionThese findings suggest that IL‐1β plays a role in exaggerating the pressor response to muscle contraction and tendon stretch in T2DM rats.

Exaggerated Activities of P2X3 and ASIC3 Signaling Pathway in Muscle Afferent Following Himblimb Muscle Ischemia‐Reperfusion

BackgroundPeripheral artery disease (PAD) is one of the major cardiovascular concerns and dramatically reduces the blood flow supply in the affected limbs. A combination of revascularization surgery in the femoral artery and supervised exercise intervention is among the most general approaches for the treatment of PAD. The approach of revascularization inevitably induces ischemia‐reperfusion (IR) injury in the tissues with the reperfusion of blood flow. Notably, following muscle IR in rats, we have observed an exaggerated exercise pressor reflex (EPR) indicating a risk of cardiovascular diseases, as the increased blood pressure (BP) response during exercise. In specific, a time‐course study (18, 66 and 114 hours post‐IR) showed that an increment in BP response was most profound in 18 hours post‐IR (IR 18h) among all the time points. However the underlying molecular mechanism leading to the IR‐induced exaggerated EPR has yet poorly understood.AimsWe targeted if the hindlimb muscle IR alters the activities of two key signaling pathway in muscle afferent nerves in regulating the EPR, namely P2X3 and ASIC3.MethodsWestern blot technique was used to determine the protein expression of P2X3 and ASIC3 in L4‐6 dorsal root ganglion (DRG) of sham rats and IR 18h rats. Meanwhile, the mean arterial pressure (MAP) responses to α‐β‐me‐ATP and lactic acid intra‐arterially injected into the hindlimb were examined to assess the P2X3 and ASIC3‐mediated EPR, respectively. Whole‐cell patch clamp was applied to evaluate the P2X and ASIC3 currents in the isolated muscle DRG neurons in both sham and IR 18h rats. All data were presented as mean ± standard deviation.ResultsCompared with sham rats, there were significant increases in both P2X3 and ASIC3 protein expression in L4‐6 DRGs in IR 18h rats (P2X3: 1.56±0.10 in IR 18h rats/n=4 vs. 1.00±0.10 in sham rats/n=6; P&lt;0.05; and ASIC3: 1.42±0.47 in IR 18h rats/n=8 vs. 1.00±0.07 in sham rats/n=10; P&lt;0.05). In addition, compared with sham rats, the MAP responses following the injection of α‐β‐me‐ATP (0.125 mM) and lactic acid (2 and 4 µmol/kg) were significantly amplified in IR 18h rats (α‐β‐me‐ATP: 29±8 mmHg in IR 18h rats/n=5 vs. 20±5 mmHg in sham rats/n=2; P&lt;0.05; Lactic acid: 2 µmol/kg, 28±8 mmHg in IR 18h rats/n=15 vs. 21±7 mmH in sham rats/n=8, P&lt;0.05; 4 µmol/kg, 37±10 mmHg in IR 18h rats/n=15 vs. 25±6 mmH in sham rats/n=9, P&lt;0.01). In the isolated DRG neurons, amplitude of both P2X and ASIC3 currents was significantly higher in IR 18h rats than that in sham rats. i.e., P2X transient currents: 1281.64±776.74 pA in IR 18h rats/n=25, vs. 717.91±556.2 pA in sham rats/n=9, (P&lt;0.05); and sustain currents: 466.94±168.68 pA in IR 18h rats/n=11, vs. 271.49±200.90 pA in sham rats/n=8 (P&lt;0.05).ConclusionThe activities of the P2X3 and ASIC3 signaling pathways in the muscle afferents are exaggerated following muscle IR. Results of this study shed light upon the future potential intervention strategy to minimize the IR‐induced exaggerated EPR response in the post‐revascularization PAD patients.

Exposure to Repeated Cold Stress Influences Sympathetic and Cardiovascular Responses to Muscle Stretch in Decerebrated Rats

Chronic stress is known to cause adverse physical and mental effects such as pain, chronic fatigue, and depression, and it is strongly related to many diseases and syndromes (e.g., fibromyalgia, chronic fatigue syndrome, and post‐traumatic stress disorder). Evidence suggests that exercise and physical activity in leisure time are effective in improving stress‐induced symptoms. Repeated cold stress (RCS), in which an animal is repeatedly exposed to alternating room and low temperatures, is one of the chronic stress models that induces chronic pain and depression. Mechanical hyperalgesia through thin muscle afferent fibers has also been reported to occur from RCS exposure. Since the muscle afferents have a dual modulatory function in nociception and cardiovascular reflex, we hypothesized that RCS augments the mechanical component of the exercise pressor reflex (the skeletal muscle mechanoreflex), i.e., skeletal muscle afferents‐mediated increases sympathetic nerve activity (SNA) and arterial blood pressure (AP).[Purpose]The purpose of this study was to clarify the impacts of RCS on sympathetic and cardiovascular responses to stimulation of the skeletal muscle mechanoreflex in decerebrated rats.[Methods]Male Sprague‐Dawley rats (body weight: 410 ± 15 g, age: 12 weeks) were exposed to RCS using a homemade automated RCS device. The rats were alternately moved to room temperature (22°C) and cold temperature (4°C) compartments at 30‐min intervals for 5 days. To assess the skeletal muscle mechanoreflex function, we measured mean AP (MAP), heart rate (HR), and renal SNA (RSNA) responses to 30‐s static passive stretching of the hindlimb muscles by in vivo recording from unanesthetized decerebrated rats. Stretching was performed by tracing the maximum tension curve obtained from 30‐s electrical stimulation. Changes (Δ) from baseline to peak values of the measured parameters between control and RCS rats were compared.[Results]Peak tension during the Achilles tendon stretch from baseline was not significantly different between groups (control: Δ929 ± 63 g [n = 6] vs RCS: Δ765 ± 66 g [n = 4], p = 0.17). Importantly, the peak RSNA response in RCS rats (Δ236.3% and Δ242.7%, n = 2) tended to be greater than that in control rats (Δ37.1% and Δ23.9%, n = 2). Moreover, RCS had a significantly (p &lt; 0.05) greater HR response (RCS: Δ3.7 ± 1.0 bpm [n = 4] vs control: Δ0.6 ± 0.5 bpm [n = 6]), and MAP response tended (P = 0.07) to be higher in the RCS group (Δ23 ± 6 mmHg, n = 4) than in the control group (Δ8 ± 4 mmHg, n = 6).[Conclusions]Our preliminary data demonstrate that RCS augments the skeletal muscle mechanoreflex. These results suggest that chronic stress can potentially cause exaggerated cardiovascular responses during physical activity.

On the Regulation of Regional Cerebral Blood Flow during Hypoxic and Hypercapnic Exercise in Humans

BackgroundIt has been previously documented that the circulatory interaction resulting from the co‐activation of the arterial chemoreflex and the exercise pressor reflex is different when the chemoreflex is evoked by hypoxia vs. hypercapnia. While the interaction effect on sympathoexcitation is simply additive during hypercapnic exercise, it is hyper‐additive during hypoxic exercise. We hypothesize that a relative hypoperfusion and compromised O2 delivery (DO2) to O2‐sensitive brainstem areas/cardiovascular control centers may trigger the Cushing’s mechanism and thereby account, in part, for this potentiated sympathetic activation during exercise in hypoxia. However, although hypoxia, hypercapnia, and exercise are recognized for their strong influence on the cerebrovascular control, the interaction between these stressors and the resulting impact upon regional brain perfusion remains unknown.PurposeTo evaluate the cerebrovascular consequence of the interaction of hypoxia, or hypercapnia, with exercise in humans.MethodOn separate occasions, six healthy volunteers rested and performed rhythmic handgrip exercise (1 Hz, 50% MVC, 3 min) in normoxia (NormRest or NormExercise: SpO2 ~97%, PETO2 ~83 mmHg, PETCO2 ~36 mmHg), normocapnic hypoxia (HypoRestor HypoExercise: SpO2 ~84%, PETO2 ~49 mmHg, PETCO2 ~36 mmHg), and normoxic hypercapnia (HyperRest or HyperExercise: SpO2 ~98%, PETO2 ~111 mmHg, PETCO2 ~46 mmHg). Doppler ultrasound was used to quantify blood flow in the contralateral internal carotid artery (ICA‐BF) and ipsilateral vertebral artery (VA‐BF), and DO2 to the anterior (via ICA‐BF) and posterior (via VA‐BF) brain regions.ResultsExercise alone (i.e., ΔNormExercise‐NormRest) significantly increased ICA‐BF (+52 ± 24 mL· min‐1), VA‐BF (+27 ± 13 mL ·min‐1), and anterior (+11 ± 5 mL ·min‐1) and posterior DO2 (+6 ± 3 mL· min‐1). Hypoxia alone (i.e., ΔHypoRest‐NormRest) significantly increased ICA‐BF (+54 ± 17 mL· min‐1) and VA‐BF (+15 ± 5 mL ·min‐1), but did not affect anterior and posterior DO2 (p ≥ 0.23). Hypercapnia alone (i.e., ΔHyperRest‐NormRest) significantly increased ICA‐BF (+135 ± 27 mL· min‐1), VA‐BF (+54 ± 11 mL· min‐1), and anterior (+28 ± 5 mL ·min‐1) and posterior DO2 (+11 ± 2 mL ·min‐1). During hypoxic exercise (i.e., ΔHypoExercise‐NormRest), the responses of ICA‐BF, VA‐BF, and regional DO2 were significantly lower compared to the sum of the responses elicited by each stressor alone (ICA‐BF: 48 ± 20 vs. 106 ± 37 mL ·min‐1; VA‐BF: 24 ± 8 vs. 41 ± 10 mL ·min‐1; anterior DO2: 0 ± 4 vs. 12 ± 7 mL· min‐1; posterior DO2: 1 ± 1 vs. 5 ± 2 mL· min‐1). During hypercapnic exercise (i.e., ΔHyperExercise‐NormRest), the observed cerebrovascular responses did not differ from the summated responses (p ≥ 0.12).ConclusionWhile the interaction between hypercapnia and exercise is additive in terms of regional cerebral blood flow and DO2, the interaction between hypoxia and exercise is hypo‐additive. The resulting hypoperfusion and compromised DO2 to the brainstem (i.e., VA‐BF and posterior DO2), via the Cushing’s mechanism, might therefore contribute to the potentiated sympathoexcitation previously observed during hypoxic exercise.