Fairfax ST, Padilla J, Vianna LC, Davis MJ, Fadel PJ. Spontaneous bursts of muscle sympathetic nerve activity decrease leg vascular conductance in resting humans. Am J Physiol Heart Circ Physiol 304: H759 -H766, 2013. First published January 4, 2013 doi:10.1152/ajpheart.00842.2012.-Previous studies in humans attempting to assess sympathetic vascular transduction have related large reflex-mediated increases in muscle sympathetic nerve activity (MSNA) to associated changes in limb vascular resistance. However, such procedures do not provide insight into the ability of MSNA to dynamically control vascular tone on a beat-by-beat basis. Thus we examined the influence of spontaneous MSNA bursts on leg vascular conductance (LVC) and how variations in MSNA burst pattern (single vs. multiple bursts) and burst size may affect the magnitude of the LVC response. In 11 young men, arterial blood pressure, common femoral artery blood flow, and MSNA were continuously recorded during 20 min of supine rest. Signal averaging was used to characterize percent changes in LVC for 15 cardiac cycles following heartbeats associated with and without MSNA bursts. LVC significantly decreased following MSNA bursts, reaching a nadir during the 6th cardiac cycle (single bursts, Ϫ2.9 Ϯ 1.1%; and multiple bursts, Ϫ11.0 Ϯ 1.4%; both, P Ͻ 0.001). Individual MSNA burst amplitudes and the total amplitude of consecutive bursts were related to the magnitude of peak decreases in LVC. In contrast, cardiac cycles without MSNA bursts were associated with a significant increase in LVC (ϩ3.1 Ϯ 0.5%; P Ͻ 0.001). Total vascular conductance decreased in parallel with LVC also reaching a nadir around the peak rise in arterial blood pressure following an MSNA burst. Collectively, these data are the first to assess beat-by-beat sympathetic vascular transduction in resting humans, demonstrating robust and dynamic decreases in LVC following MSNA bursts, an effect that was absent for cardiac cycles without MSNA bursts.MSNA; sympathetic vascular transduction; vascular responsiveness; blood pressure; femoral blood flow THE GENERATION AND REGULATION of central sympathetic outflow has been a primary focus of autonomic cardiovascular research for decades (5, 10). However, the ability of the peripheral vasculature to respond to changes in sympathetic activity has received less attention. Indeed, for sympathetic nerve impulses to influence vascular tone, there must be successful release of neurotransmitter(s) into the synaptic cleft (2), abundant receptor binding (35), and widespread signal transduction within vascular smooth muscle (34) to result in a vasomotor response (29). Although this paradigm is widely accepted (3,12,26), limited data are available in humans. Furthermore, previous studies attempting to demonstrate sympathetic vascular transduction in humans have related large reflex-mediated increases in muscle sympathetic nerve activity (MSNA) to associated changes in vascular resistance (6,17,23). While such procedures reveal that elevated MSNA increases vascular...

Key Points• Sympathetic support of blood pressure demands the efficient control of vascular tone; however, little is known regarding how spontaneously occurring bursts of muscle sympathetic nerve activity (MSNA) dynamically influence forearm vascular conductance.• This study examined the extent to which spontaneous MSNA bursts evoke changes in forearm vascular conductance and blood pressure with and without local α-adrenergic blockade in young healthy men during supine rest.• We observed that under resting conditions, forearm vascular conductance increases briefly and then significantly decreases in association with the total amount of the preceding MSNA; however, during α-adrenergic blockade the decrease in vascular conductance is eliminated.• These results indicate that normal variations in spontaneous MSNA burst activity are systematically followed by transient and robust responses of forearm vasoconstriction and that this influence is mediated via α-adrenergic receptor mechanisms.Abstract Sympathetic vascular transduction is commonly understood to act as a basic relay mechanism, but under basal conditions, competing dilatory signals may interact with and alter the ability of sympathetic activity to decrease vascular conductance. Thus, we determined the extent to which spontaneous bursts of muscle sympathetic nerve activity (MSNA) mediate decreases in forearm vascular conductance (FVC) and the contribution of local α-adrenergic receptor-mediated pathways to the observed FVC responses. In 19 young men, MSNA (microneurography), arterial blood pressure and brachial artery blood flow (duplex Doppler ultrasound) were continuously measured during supine rest. These measures were also recorded in seven men during intra-arterial infusions of normal saline, phentolamine (PHEN) and PHEN with angiotensin II (PHEN+ANG). The latter was used to control for increases in resting blood flow with α-adrenergic blockade. Spike-triggered averaging was used to characterize beat-by-beat changes in FVC for 15 cardiac cycles following each MSNA burst and a peak response was calculated. Following MSNA bursts, FVC initially increased by +3.3 ± 0.3% (P = 0.016) and then robustly decreased to a nadir of −5.8 ± 1.6% (P < 0.001). The magnitude of vasoconstriction appeared graded with the number of consecutive MSNA bursts; while individual burst size only had a mild influence. Neither PHEN nor PHEN+ANG infusions affected the initial rise in FVC, but both infusions significantly attenuated the subsequent decrease in FVC (-2.1 ± 0.7% and -0.7 ± 0.8%, respectively; P < 0.001 vs. normal saline). These findings indicate that spontaneous

The sympathetic nervous system is critical for the beat-to-beat regulation of arterial blood pressure (BP). Although studies have examined age- and sex-related effects on BP control, findings are inconsistent and limited data are available in postmenopausal women. In addition, the majority of studies have focused on time-averaged responses without consideration for potential beat-to-beat alterations. Thus we examined whether the ability of muscle sympathetic nerve activity (MSNA) to modulate BP on a beat-to-beat basis is affected by age or sex. BP and MSNA were measured during supine rest in 40 young (20 men) and 40 older (20 men) healthy subjects. Beat-to-beat fluctuations in mean arterial pressure (MAP) were characterized for 15 cardiac cycles after each MSNA burst using signal averaging. The rise in MAP following an MSNA burst was similar between young men and women (+2.64 ± 0.3 vs. +2.57 ± 0.3 mmHg, respectively). However, the magnitude of the increase in MAP after an MSNA burst was reduced in older compared with young subjects (P < 0.05). Moreover, the attenuation of the pressor response was greater in older women (+1.20 ± 0.1 mmHg) compared with older men (+1.72 ± 0.2 mmHg; P < 0.05). Interestingly, in all groups, MAP consistently decreased after cardiac cycles without MSNA bursts (nonbursts) with the magnitude of fall greatest in older men. In summary, healthy aging is associated with an attenuated beat-to-beat increase in BP after a spontaneous MSNA burst, and this attenuation is more pronounced in postmenopausal women. Furthermore, our nonburst findings highlight the importance of sympathetic vasoconstrictor activity to maintain beat-to-beat BP, particularly in older men.

The vasodilatory effects of insulin account for up to 40% of insulin-mediated glucose disposal; however, insulin-stimulated vasodilation is impaired in individuals with type 2 diabetes, limiting perfusion and delivery of glucose and insulin to target tissues. To determine whether exercise training improves conduit artery blood flow following glucose ingestion, a stimulus for increasing circulating insulin, we assessed femoral blood flow (FBF; Doppler ultrasound) during an oral glucose tolerance test (OGTT; 75 g glucose) in 11 overweight or obese (body mass index, 34 ± 1 kg/m²), sedentary (peak oxygen consumption, 23 ± 1 ml·kg⁻¹·min⁻¹) individuals (53 ± 2 yr) with non-insulin-dependent type 2 diabetes (HbA1c, 6.63 ± 0.18%) before and after 7 days of supervised treadmill and cycling exercise (60 min/day, 60-75% heart rate reserve). Fasting glucose, insulin, and FBF were not significantly different after 7 days of exercise, nor were glucose or insulin responses to the OGTT. However, estimates of whole body insulin sensitivity (Matsuda insulin sensitivity index) increased (P < 0.05). Before exercise training, FBF did not change significantly during the OGTT (1 ± 7, -7 ± 5, 0 ± 6, and 0 ± 5% of fasting FBF at 75, 90, 105, and 120 min, respectively). In contrast, after exercise training, FBF increased by 33 ± 9, 39 ± 14, 34 ± 7, and 48 ± 18% above fasting levels at 75, 90, 105, and 120 min, respectively (P < 0.05 vs. corresponding preexercise time points). Additionally, postprandial glucose responses to a standardized breakfast meal consumed under "free-living" conditions decreased during the final 3 days of exercise (P < 0.05). In conclusion, 7 days of aerobic exercise training improves conduit artery blood flow during an OGTT in individuals with type 2 diabetes.

Mauban JR, Fairfax ST, Rizzo MA, Zhang J, Wier WG. A method for noninvasive longitudinal measurements of [Ca 2ϩ ] in arterioles of hypertensive optical biosensor mice. Am J Physiol Heart Circ Physiol 307: H173-H181, 2014. First published May 23, 2014 doi:10.1152/ajpheart.00182.2014.-We used two-photon (2-p) Förster resonance energy transfer (FRET) microscopy to provide serial, noninvasive measurements of [Ca 2ϩ ] in arterioles of living "biosensor" mice. These express a genetically encoded Ca 2ϩ indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. The FRET ratios, Rmin and Rmax, required for in vivo Ca 2ϩ calibration of exMLCK were obtained in isolated arteries. For in vivo experiments, mice were anesthetized (1.5% isoflurane), and arterioles within a depilated ear were visualized through the intact skin (i.e., noninvasively), by 2-p excitation of exMLCK (at 820 nm) or GCaMP2 (at 920 nm). Spontaneous or agonist-evoked [Ca 2ϩ ] transients in arteriolar smooth muscle cells were imaged (at 2 Hz) with both exMLCK and GCaMP2. To examine changes in arteriolar [Ca 2ϩ ] that might accompany hypertension, five exMLCK mice were implanted with telemetric blood pressure transducers and osmotic minipumps containing ANG II (350 ng·kg Ϫ1 ·min Ϫ1 ) and fed a high (6%)-salt diet for 9 days. [Ca 2ϩ ] was measured every other day in five smooth muscle cells of two to three arterioles in each animal. Prior to ANG II/salt, [Ca 2ϩ ] was 246 Ϯ 42 nM. [Ca 2ϩ ] increased transiently to 599 nM on day 2 after beginning ANG II/salt, then remained elevated at 331 Ϯ 42 nM for 4 more days, before returning to 265 Ϯ 47 nM 6 days after removal of ANG II/salt. In summary, two-photon excitation of exMLCK and GCaMP2 provides a method for noninvasive, longitudinal quantification of [Ca 2ϩ ] dynamics and vascular structure in individual arterioles of a particular animal over an extended period of time, a capability that should enhance future studies of hypertension and vascular function.calcium; FRET; hypertension; two photon; vascular STUDIES of experimental hypertension in animals are confronted with several fundamental technical difficulties. Determining how changes in arterial [Ca 2ϩ ] might be involved in experimental hypertension is particularly challenging because measurements of [Ca 2ϩ ] and artery contractile activation will change immediately upon removal of an artery for study. The processes controlling [Ca 2ϩ

Large increases in muscle sympathetic nerve activity (MSNA) can decrease the diameter of a conduit artery even in the presence of elevated blood pressure, suggesting that MSNA acts to regulate conduit artery tone. Whether this influence can be extrapolated to spontaneously occurring MSNA bursts has not been examined. Therefore, we tested the hypothesis that MSNA bursts decrease conduit artery diameter on a beat-by-beat basis during rest. Conduit artery responses were assessed in the brachial (BA), common femoral (CFA) and popliteal (PA) arteries to account for regional differences in vascular function. In 20 young men, MSNA, mean arterial pressure (MAP), conduit artery diameter, and shear rate (SR) were continuously measured during 20-min periods of supine rest. Spike-triggered averaging was used to characterize beat-by-beat changes in each variable for 15 cardiac cycles following all MSNA bursts, and a peak response was calculated. Diameter increased to a similar peak among the BA (+0.14 ± 0.02%), CFA (+0.17 ± 0.03%), and PA (+0.18 ± 0.03%) following MSNA bursts (all P < 0.05 vs. control). The diameter rise was positively associated with an increase in MAP in relation to increasing amplitude and consecutive numbers of MSNA bursts (P < 0.05). Such relationships were similar between arteries. SR changes following MSNA bursts were heterogeneous between arteries and did not appear to systematically alter diameter responses. Thus, in contrast to our hypothesis, spontaneously occurring MSNA bursts do not directly influence conduit arteries with local vasoconstriction or changes in shear, but rather induce a systemic pressor response that appears to passively increase conduit artery diameter.

Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction.

Investigations of human myogenic responses typically use maneuvers that evoke robust changes in transmural pressure. Although this strategy has demonstrated peripheral myogenic responsiveness in the limbs, particularly in glabrous skin of the hand or foot, it has not considered the potential influence of the myogenic mechanism in beat-to-beat blood flow (BF) control during unprovoked rest. In the present study, we examined the interactions of spontaneous beat-to-beat mean arterial pressure (MAP; Finapres) with BF (Doppler ultrasound) supplying the forearm (brachial artery), lower leg (popliteal artery), and hand (ulnar artery) during 10 min of supine rest in healthy young men. Cross-correlation analyses revealed a negative association between MAP and BF, which was more prominent in the forearm than lower leg. The strongest correlation resulted when a -2-heart beat offset of MAP was applied (R=-0.53±0.04 in the forearm and -0.23±0.05 in the leg, P<0.05), suggesting an ∼2-s delay from instances of high/low MAP to low/high BF. Negatively associated episodes (high MAP/low BF and low MAP/high BF) outnumbered positively associated data (P<0.05). BF during low MAP values was greater than the steady-state average BF and vice versa. Wrist and ankle occlusion blunted the strength of correlations, homogenized the incidence of MAP and BF pairings, and reduced the magnitude of deviation from steady-state values. In contrast, these relationships were matched or accentuated for hand BF. Overall, these results suggest that myogenic responses are present and occur rapidly in human limbs during rest, overwhelm perfusion pressure gradient influences, and are primarily mediated by the distal limb circulation.