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Effect of cold exposure and shear stree on endothelial nitric oxide synthase activation
Abstract
Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37°C and 28°C, and a decrease at 4°C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.
... Several hypotheses on the physiological mechanism of CIVD have been proposed. It has been suggested that CIVD is caused by neurogenic activity, such as the cessation of adrenergic neurotransmission, the release of dilating substances [e.g., nitric oxide (NO)] in the endothelium of blood vessels, and myogenic activity, such as the relaxation of the smooth muscle cells [2,[6][7][8]. Our previous study revealed that both neurogenic activity and endothelial NO (eNO)-independent activity may affect the finger SkBF response during immersion in cold water at 5 °C [9]. ...
Background
Cold-induced vasodilation (CIVD) occurs after blood vessels in the skin are constricted due to local cold exposure. Although many CIVD studies have been conducted, the underlying molecular mechanisms are yet to be clarified. Therefore, we explored genetic variants associated with CIVD response using the largest-scale dataset reported to date in a CIVD study involving wavelet analysis; thus, the findings improve our understanding of the molecular mechanisms that regulate the CIVD response.
Methods
We performed wavelet analysis of three skin blood flow signals [endothelial nitric oxide (eNO)-independent, eNO-dependent, and neurogenic activities] during finger cold-water immersion at 5 °C in 94 Japanese young adults. Additionally, we conducted genome-wide association studies of CIVD using saliva samples collected from the participants.
Results
We found that the mean wavelet amplitudes of eNO-independent and neurogenic activities significantly increased and decreased prior to CIVD, respectively. Our results also implied that as many as ~ 10% of the Japanese subjects did not show an apparent CIVD response. Our genome-wide association studies of CIVD using ~ 4,040,000 imputed data found no apparent CIVD-related genetic variants; however, we identified 10 genetic variants, including 2 functional genes ( COL4A2 and PRLR ) that are associated with notable blunted eNO-independent and neurogenic activity responses in individuals without CIVD response during local cold exposure.
Conclusions
Our findings indicate that individuals without CIVD response differentiated by genotypes with COL4A2 and PRLR genetic variants exhibited notable blunted eNO-independent and neurogenic activity responses during local cold exposure.
... Exercise-induced shear stress combined with cold temperature may lead to impaired regulation of vascular tonus [36,46] and activation of vWF [33,34] which further potentiates blood coagulation. Moreover, atherosclerosis and CAD itself could result in elevated coagulation potential [23]. ...
Background
Both exercise and cold exposure increase blood coagulation potential but their combined effects are not known. The purpose of the present study was to assess blood coagulation factors in response to submaximal exercise in the cold environment among patients with stable coronary artery disease (CAD).
Methods
Sixteen men (61.1 ± 7.1 years) with stable CAD participated in three 30-min experimental conditions (seated rest in − 15 °C and exercise in both + 22 °C and − 15 °C) in random order. The employed exercise consisted of brisk walking (66–69% of maximal heart rate). Factor VII (FVII), fibrinogen, D-dimer and von Willebrand factor (vWF) were analyzed from blood samples obtained before, immediately and one hour after each experiment.
Results
On average, FVII activity (95% confidence interval, CI) was 123 (108–143) %, 123 (106–140) %, 121 (103–139) % (baseline, recovery 1, recovery 2), fibrinogen concentration (95% CI) 3.81 (3.49–4.12) g/l, 3.71 (3.34–4.08) g/l, 3.65 (3.26–4.05) g/l, D-dimer concentration (95% CI) 0.42 (0.28–0.56) µg/ml, 0.42 (0.29-.55) µg/ml and 0.39 (0.29–0.49) µg/ml, and vWF activity (95% CI) 184 (135–232) %, 170 (128–212) % and 173 (129–217) % after exercise in the cold. Average FVII activity varied from 122 to 123%, fibrinogen concentration from 3.71 to 3.75 g/l, D-dimer concentration from 0.35 to 0.51 µg/ml and von Willebrand factor activity from 168 to 175% immediately after each three experimental condition.
Conclusions
Our findings suggest that submaximal lower body exercise carried out in a cold environment does not significantly affect blood coagulation parameters among patients with stable CAD.
... This is likely due to differential effects on ECs from different ages, vascular beds and potentially to both the duration and level of the mechanical force utilized [43,[45][46][47]. In addition, although laminar shear stress predominantly stimulates eNOS activity and NO release [48][49][50][51][52], oscillatory flow uncouples eNOS [53]. Thus, different types of mechanical forces can act differently on regions of the vascular wall to affect NO bioavailability and potentially contribute to disease pathogenesis. ...
Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.
... AMPK (57,58). AMPK is known to phosphorylate the specific serine residue (Ser1177) of eNOS that is essential for its activation (58,59). ...
Aged garlic extract (AGE) has been shown to improve peripheral circulatory disturbances in both clinical trials and experimental animal models. To investigate the effect of S-1-propenylcysteine (S1PC), a characteristic sulfur compound in AGE, on cold-induced reduction in tail blood flow of rat, Wistar rats were individually placed in a restraint cage and given the treatment with cold water (15˚C) after the oral administration of AGE or its constituents S1PC, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC). After the cold-treatment the tail blood flow of rats was measured at the indicated times. The pretreatment with AGE (2 g/kg BW) and S1PC (6.5 mg/kg BW) significantly alleviated the reduction of rat tail blood flow induced by cold treatment. The effect of S1PC was dose-dependent and maximal at the dose of 6.5 mg/kg BW, whereas SAC and SAMC were ineffective. To gain insight into the mechanism of S1PC action, the concentration of nitrogen oxide metabolites (NOx) in the plasma and the levels of phosphorylated endothelial nitric oxide synthase (eNOS) and 5'-AMP-activated protein kinase (AMPK) in the aorta were measured. The pretreatment with S1PC significantly increased the plasma concentration of NOx as well as the level of phosphorylated form of AMPK and eNOS in the aorta after cold-treatment. The present findings suggest that S1PC is a major constituent responsible for the effect of AGE to alleviate the cold-induced reduction of peripheral blood flow in rat by acting on the AMPK/eNOS/NO pathway in the aorta.
... While this is possible, CIVD may also be an active process mediated by non-adrenergic metabolites. Chief among these candidates is the potent vasodilator nitric oxide (Johnson and Kellogg 2010); however, data to date are equivocal and inconclusive due to a combination of indirect assessments of nitric oxide production and endothelial nitric oxide synthase activity (Therminarias et al. 1998;Binti Md Isa et al. 2011;Stensrud et al. 2016;Hodges et al. 2006). Ultimately, whether the acute increase in blood flow and temperature despite sustained cold ambient temperatures is due to withdrawal of sympathetic function or an active process elicited by some other metabolic or hormonal mediator (e.g., nitric oxide) is unclear (Cheung and Daanen 2012;Cheung 2009, 2010;Sessler 2009). ...
Whether sympathetic withdrawal or endothelial dilators such as nitric oxide (NO) contributes to cold-induced vasodilation (CIVD) events is unclear. We measured blood flow and finger skin temperature (Tfinger) of the index finger in nine participants during hand immersion in a water bath at 35 °C for 30 min, then at 8 °C for 30 min. Data were binned into 10 s averages for the entire 60 min protocol for laser-Doppler flux (LDF) and Tfinger. At baseline, Tfingerwas 35.3 ± 0.2 °C and LDF was 227 ± 28 PU. During hand cooling, minimum Tfingerwas 10.9 ± 0.4 °C and LDF was 15 ± 4 PU. All participants exhibited at least one CIVD event (Tfingerincrease ≥ 1 °C), with a mean peak Tfinger13.2 ± 0.8 °C and a corresponding peak LDF of 116 ± 34 PU. A Morlet mother wavelet was then used to perform wavelet analysis on the LDF signal, with frequency ranges of 0.005-0.01 Hz (endothelial NO-independent), 0.01-0.02 Hz (endothelial NO-dependent), and 0.02-0.05 Hz (neurogenic). The synchronicity of wavelet fluctuations with rising LDF coincident with CIVD events was then quantified using Auto-regressive Integrated Moving Average time-series analysis. Fluctuations in neural activity were strongly synchronized in real time with increasing LDF (stationary-r2 = 0.73 and Ljung-box statistic > 0.05), while endothelial activities were only moderately synchronized (NO-independent r2 = 0.15, > 0.05; NO dependent r2 = 0.16, > 0.05). We conclude that there is a direct, real-time correlation of LDF responses with neural activity but not endothelial-mediated mechanisms. Importantly, it seems that neural activity is consistently reduced prior to CIVD, suggesting that sympathetic withdrawal directly contributes to CIVD onset.
... Animal experiments have demonstrated rise in oxygen consumption and ventilation in hypoxic conditions on cold exposure [14,15] (number of cases with HAPE were more in winter as compared to summer). Evidence is also suggestive of altered nitric oxide dynamics in pulmonary endothelium [16] on cold exposure increasing the likelihood of HAPE. Decreased NO levelshavebeenattributed by authors [17] toward increased leukocyte adherence due to hypoxia in animals experiments conducted on endothelium in other tissues. ...
Objectives: To evaluate the safety and efficacy of treating high-altitude pulmonary edema (HAPE) by bed rest and supplemental oxygen in hospital setting at high altitude. Materials and Methods: In a prospective case series, all patients who were diagnosed clinically with HAPE on admission to our hospital located at a height of 11,500 ft were evaluated and managed with bed rest and oxygen supplementation. Results: A total of 43 patients of HAPE with mean age of 31 years (range 20–48 years) were admitted to our hospital. Infections followed by unaccustomed physical exertion were the predominant risk factors. 95.35% of the patients improved successfully with oxygen and bed rest alone with mean hospital stay of 2.67 ± 1.06 (1–6 days). Two patients (4.65%) required nifedipine and evacuation to lower altitude. Of this, one patient suffering from concomitant viral infection expired 4 days after evacuation to near sea level. Conclusion: Majority of the patients with HAPE where medical facilities are available can be safely treated with bed rest and oxygen supplementation at moderate high altitude without descent.
... While it is unclear whether exhaled NO is representative or a direct result of vascular production, 126 exhaled NO during cold (¡10 C) running exercise was less than in temperate conditions both during 127 and after 128 exercise. Using in vitro microvascular endothelial cell cultures, Binti MD Isa et al. 129 tested the effects of temperature changes along with shear stress on endothelial NO synthase (eNOS) activation. Cold (4 C) temperature by itself did not affect eNOS activation over 1 h of exposure; cold combined with shear stress did not affect eNOS over 15 and 30 min, but significantly decreased its activation at 60 min. ...
An initial response to whole-body or local exposure of the extremities to cold is a strong vasoconstriction, leading to a rapid decrease in hand and foot temperature. This impairs tactile sensitivity, manual dexterity, and muscle contractile characteristics while increasing pain and sympathetic drive, decreasing gross motor function, occupational performance, and survival. A paradoxical and cyclical vasodilatation often occurs in the fingers, toes, and face, and this has been termed the hunting response or cold-induced vasodilatation (CIVD). Despite being described almost a century ago, the mechanisms of CIVD are still disputed; research in this area has remained largely descriptive in nature. Recent research into CIVD has brought increased standardization of methodology along with new knowledge about the impact of mediating factors such as hypoxia and physical fitness. Increasing mechanistic analysis of CIVD has also emerged along with improved modeling and prediction of CIVD responses. The present review will survey work conducted during this century on CIVD, its potential mechanisms and modeling, and also the broader context of manual function in cold conditions.
... For example, the laboratory-based components of the studies reviewed were performed between 22°C and 25°C, a temperature likely to be far warmer that experiences at 4,000 m in a field laboratory . Such differences in ambient temperature may alter physiological mechanisms such as the degree of peripheral vasoconstriction, NO metabolism or the production of reactive oxidative species [24]. As highlighted, the duration of the hypoxic exposure impacts on the results obtained. ...
Much hypoxia research has been carried out at high altitude in a hypobaric hypoxia (HH) environment. Many research teams seek to replicate high-altitude conditions at lower altitudes in either hypobaric hypoxic conditions or normobaric hypoxic (NH) laboratories. Implicit in this approach is the assumption that the only relevant condition that differs between these settings is the partial pressure of oxygen (PO2), which is commonly presumed to be the principal physiological stimulus to adaptation at high altitude. This systematic review is the first to present an overview of the current available literature regarding crossover studies relating to the different effects of HH and NH on human physiology. After applying our inclusion and exclusion criteria, 13 studies were deemed eligible for inclusion. Several studies reported a number of variables (e.g. minute ventilation and NO levels) that were different between the two conditions, lending support to the notion that true physiological difference is indeed present. However, the presence of confounding factors such as time spent in hypoxia, temperature, and humidity, and the limited statistical power due to small sample sizes, limit the conclusions that can be drawn from these findings. Standardisation of the study methods and reporting may aid interpretation of future studies and thereby improve the quality of data in this area. This is important to improve the quality of data that is used for improving the understanding of hypoxia tolerance, both at altitude and in the clinical setting.
... 29 All the above suggests that heat shock and low temperature can both activate the same pathway to induce the eNOS activation accordingly. 30 Thus, long term application of low shear stresses leads to generation of a pro-inflammatory environment, which may also explain the thrombosis in venous valve regions with low blood flow. ...
Current vascular grafts have a high incidence of failure, especially in the grafts less than 6 mm in diameter, due to thrombus formation. Nitric oxide (NO) is released by endothelium and has some beneficial influences such as an antithrombotic effect. We hypothesized that applying different shear stress regiments and low temperature or aspirin would result in an increase of the amount of NO release from Human umbilical vein endothelial cells (HUVECs) and decrease in platelet aggregation in the same manner as expected in vivo. HUVECs were cultured into the intraluminal surface of silicone tubes. HUVECs were subjected for 60 minutes to different paremeters of shear stress, temperature, aspirin, and platelets or a combination in a perfusion bioreactor by monitoring NO secretion. We found that shear stress leads to an elevation of NO production in HUVECS, independent of the shear stress magnitude (0.9 or 1.8 dyne/cm2). The magnitude of this response increased with a decrease in temperature.Our results also show that by addition of platelets in combination with aspirin to media circulation, no thrombus formation occurred during the test time. Presence of aspirin resulted in marked increase of NO levels. In conclusion, shear stresses, temperature lowering, and aspirin increase the amount of NO release from HUVECs. Also no thrombus formation was detected in our experimental setting.
... Although evidence exists regarding the beneficial effects of the release of small amounts of NeO during the inhibition of nociceptive pathways (Luo and Cizkova, 2000), other research suggests that NeO could contribute to hyperalgesia (Aley et al., 1998). Physiological mechanical stimuli such as blood shear stress (Binti et al., 2011) or tissue stretch (Zhang et al., 2004) induce the liberation of NeO from endothelial tissue and blood cells (Baskurt et al., 2011). It has been proven that the mechanical stimulus provided by physical therapy techniques, such as massage (Morhenn et al., 2012) or acupuncture (Tsuchiya et al., 2007), modulates pain perception by modifying Ne O production. ...
Previous studies have analyzed the effects of spinal manipulation on pain sensitivity by using several sensory modalities, but to our knowledge, no studies have focused on serum biomarkers involved in the nociceptive pathway after spinal manipulation. Our objectives were to determine the immediate effect of cervical and dorsal manipulation over the production of nitric oxide and substance P, and establishing their relationship with changes in pressure pain thresholds in asymptomatic subjects. In this single-blind randomized controlled trial, 30 asymptomatic subjects (16 men) were randomly distributed into 3 groups (n=10 per group): control, cervical and dorsal manipulation groups. Blood samples were extracted to obtain serum. ELISA assay for substance P and chemiluminiscence analysis for nitric oxide determination were performed. Pressure pain thresholds were measured with a pressure algometer at the C5-C6 joint, the lateral epicondyle and the tibialis anterior muscle. Outcome measures were obtained before intervention, just after intervention and 2 hours after intervention. Our results indicated an increase in substance P plasma level in the cervical manipulation group (70.55 %) when compared with other groups (P<0.05). This group also showed an elevation in the pressure pain threshold at C5-C6 (26.75 %) and lateral epicondyle level (21.63 %) immediately after the intervention (P<0.05). No changes in nitric oxide production were observed. In conclusion, mechanical stimulus provided by cervical manipulation increases substance P levels and pressure pain threshold but does not change nitric oxide concentrations. Part of the hypoalgesic effect of spinal manipulation may be due to the action of substance P.
... 248 NOS3 is found mainly in endothelial cells. 249 NOS2, which is also called inducible NOS (iNOS), is expressed by variety of immunological cells upon activation by IFN-γ, TNF-α, and specific chemokines. [250][251][252][253] The increased levels of these cytokines and chemokines in Gaucher disease (Table 1) and Gba1 mutant mice may underlay an increased expression of the NOS2 gene, 26 and NO and ROS proteins in the brain of Gba1 knockout mice. ...
The macrophage (MΦ) has been the focus of causality, research, and therapy of Gaucher disease, but recent evidence casts doubt its solitary role in the disease pathogenesis. The excess of glucosylceramide (GC) in such cells accounts for some of the disease manifestations. Evidence of increased expression of C-C and C-X-C chemokines (i.e., CCL2,CXCL1, CXCL8) in Gaucher disease could be critical for monocyte transformation to inflammatory subsets of macrophages and dendritic cells (DC) as well as neutrophil (PMNs) recruitment to visceral organs. These immune responses could be essential for activation of T- and B-cell subsets, and the induction of numerous cytokines and chemokines that participate in the initiation and propagation of the molecular pathogenesis of Gaucher disease. The association of Gaucher disease with a variety of cellular and humoral immune responses is reviewed here to provide a potential foundation for expanding the complex pathophysiology of Gaucher disease.
Efficiently removing blood from the brain vasculature is critical to evaluate accurately brain penetration and biodistribution of drug candidates, especially for biologics as their blood concentrations are substantially higher than the brain concentrations. Transcardial perfusion has been widely used to remove residue blood in brain; however, perfusion conditions (such as perfusion rate and time) reported in the literature are quite varied, and performance of these methods on blood removal has not been thoroughly investigated. In this study, the effectiveness of the perfusion conditions was assessed by measuring brain hemoglobin levels. Sodium nitrite (NaNO2 ) as an additive in the perfusate was evaluated at different concentrations. Blood removal was significantly improved with 2% NaNO2 over a 20-min perfusion in mouse without disrupting blood-brain barrier (BBB) integrity. In mice, the optimized perfusion method significantly lowered measured brain-to-plasma ratio (Kp,brain ) for monoclonal antibodies due to the removal of blood contamination and small molecules with moderate-to-high BBB permeability and with high brain-unbound-fraction (fu,brain ) presumably due to flux out of the brain during perfusion. Perfusion with or without NaNO2 clearly removed the residual blood in rat brain but with no difference observed in Kp,brain between perfusion groups with or without 2% NaNO2 . In conclusion, the perfusion method was successfully developed to evaluate the brain penetration of small molecules and biologics in rodents for the first time. The transcardial perfusion with 2% NaNO2 effectively removed the residual blood in the brain and significantly improved the brain penetration assessment for biologics. For small molecules, however, transcardial perfusion may not be performed, as small molecule compounds could be washed away from the brain by the perfusion procedure. This article is protected by copyright. All rights reserved.
Growing evidence has shown that exposure to low ambient temperature poses a huge challenge to human health globally. Actually, cold stress is closely associated with a higher incidence of cardiovascular morbidity and mortality in winter or in cold regions. Cellular and molecular mechanisms underlying cardiovascular complications in response to cold exposure have yet to be fully clarified. Considering that cold exposure is an important risk of cardiovascular complications, it is necessary to clarify the molecular mechanism of cold stress-induced cardiovascular diseases and to develop effective intervention strategies. Hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) are well-known gasotransmitters that are endogenously produced in many biological systems. Accumulating studies have demonstrated that these gasotransmitters play a critical role in a wide spectrum of physiological and/or pathophysiological processes by regulating numerous signaling pathways. These gas signal molecules are emerging as important players in cardiovascular homeostasis, and disruption of these gasotransmitters is critically implicated in cardiovascular anomalies, such as hypertension, atherosclerosis, myocardial ischemia, heart failure, and stroke. Also, evidence is emerging that H2S, NO, and CO may be involved in the pathologies of cold stress-induced cardiovascular ailments. In this review, we aim to highlight and discuss the recent advances towards the development of gasotransmitters-based therapeutics for cold stress-related cardiovascular pathogenesis. We believe that the effects of H2S, NO, and CO on cardiovascular regulation under cold environment will attract tremendous interest in the near future as they serve as novel regulators of cardiovascular biology in cold environment.
PurposeThis study investigated whether muscle cooling and its associated effects on skeletal muscle oxidative responses, blood gases, and hormonal concentrations influenced energy metabolism during cycling.Methods
Twelve healthy participants (Males: seven; Females: five) performed two steady-state exercise sessions at 70% of ventilatory threshold on a cycle ergometer. Participants completed one session with pre-exercise leg cooling until muscle temperature (Tm) decreased by 6 °C (LCO), and a separate session without cooling (CON). They exercised until Tm returned to baseline and for an additional 30 min. Cardiovascular, respiratory, metabolic, hemodynamic variables, and skeletal muscle tissue oxidative responses were assessed continuously. Venous blood samples were collected to assess blood gases, and hormones.ResultsHeart rate, stroke volume, and cardiac output all increased across time but were not different between conditions. V̇O2 was greater in LCO when muscle temperature was restored until the end of exercise (p < 0.05). Cycling in the LCO condition induced lower oxygen availability, tissue oxygenation, blood pH, sO2%, and pO2 (p < 0.05). Insulin concentrations were also higher in LCO vs. CON (p < 0.05). Importantly, stoichiometric equations from respiratory gases indicated no differences in fat and CHO oxidation between conditions.Conclusion
The present study demonstrated that despite muscle cooling and the associated oxidative and biochemical changes, energy metabolism remained unaltered during cycling. Whether lower local and systemic oxygen availability is counteracted via a cold-induced activation of lipid metabolism pathways needs to be further investigated.
Chronic exposure to cold causes arterial hypertension (CIH). Emerging data have indicted that gut barrier dysfunction is involved in the pathogenesis of hypertension. In this study we explored the effect of gut barrier dysfunction on vascular inflammation induced by cold exposure and the therapeutic effect of atorvastatin in a CIH rat model. The CIH was established by cold exposure for two weeks. Two groups of Sprague Dawley (SD) rats were exposed to moderate cold (4 ± 1 °C), while the control group was maintained at room temperature (RT) (23 ± 1 °C) (10 rats/group). The two groups were received atorvastatin or vehicle at beginning of cold exposure, respectively, for two weeks. Cold exposure increased mean arterial pressure (MAP) compared to RT group, indicating that animals developed arterial hypertension. Cold exposure induced vascular dysfunction due to decreasing phosphorylated eNOS protein expression in aorta, and these were blunted by atorvastatin. Cold exposure increased the levels of gut-derived inflammatory cytokines, TNF-α and IL-6 production in aorta, and resulted in vascular inflammation, while atorvastatin prevented these effects. Cold exposure also increased gut permeability, inhibited tight junction protein expression in proximal colon and resulted in gut barrier dysfunction. Interestingly, atorvastatin eliminated increasing of gut permeability, decreasing of tight junction protein expression, gut pathology and reversed gut barrier dysfunction. Atorvastatin attenuated CIH and improved gut barrier function, the beneficial effects might be via inhibiting gut-derived inflammatory cytokines and reversing cold-induced vascular inflammation, suggesting that gut barrier dysfunction may be involved in the pathogenesis of CIH.
Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.
Seasonal variation of flow-mediated vasodilation (FMD) remains controversial. A large cohort study showing that FMD was highest in summer and lowest in winter has been performed in a cross-sectional manner on different populations in different seasons, and the results for the same population were not compared.
FMD was compared between the cool season (14.4 ± 4.4°C) and warm season (28.8 ± 1.0°C) in the same 27 outpatients with hypertension, diabetes mellitus and/or hyperlipidemia.
The mean resting brachial artery diameter was significantly larger in the warm season than in the cool season. The maximal post-deflation brachial artery diameter was also significantly larger in the warm season than in the cool season. FMD, which was calculated from the resting diameter and the maximal diameter, was significantly higher in the warm season than in the cool season even when expressed as the relative value (4.74 ± 2.15 vs. 5.71 ± 2.17%, p=0.03) or absolute value (0.18 ± 0.08 vs, 0.23 ± 0.07 mm, p=0.008).
FMD was significantly higher in the warm season than in the cool season when the measurements were performed on the same subjects and a paired comparison was made.
During exercise, ischemic risk increases, possibly due to changes in coagulation and fibrinolytic activity. Previous research suggests ambient temperature affects resting thrombotic potential, but the effect of heat and cold on hemostasis during exercise is unknown. The purpose of this study was to assess changes in coagulation and fibrinolysis during maximal exercise in hot and cold temperatures, and to compare those responses to exercise under temperate conditions.
Fifteen healthy men completed maximal exercise tests in hot (30°C), temperate (20°C) and cold (5° - 8°C) temperatures. Blood samples were obtained before and immediately after exercise and analyzed for concentrations of thrombin-antithrombin III (TAT), active tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Results were analyzed by ANOVA.
A main effect of time was observed for TAT (temperate=1.71 ± 0.82 - 2.61 ± 0.43 ng/ml, hot=1.81 ± 0.73 - 2.62 ± 0.67 ng/ml, cold=2.33 ± 0.65 - 2.89 ± 0.81 ng/ml, PRE to POST, respectively) and tPA activity (temperate=0.72 ± 0.44 - 2.71 ± 0.55 IU/ml, hot=0.72 ± 0.38 - 2.64 ± 0.61 IU/ml, cold=0.86 ± 0.45 - 2.65 ± 0.77 IU/ml, PRE to POST, respectively). A trend was observed for the PAI-1 response to exercise (temperate=14.5 ± 23.7 - 12.3 ± 20.2I U/ml, hot=15.1 ± 26.5 - 10.0 ± 15.1 IU/ml, cold=10.5 ± 10.4 - 7.9 ± 9.7 IU/ml, PRE to POST, respectively, p=0.08). TAT concentrations were significantly higher in cold compared to temperate and hot conditions.
Coagulation potential is elevated during exposure to cold temperatures. These data suggest that risk of an ischemic event may be elevated in the cold.
ture that clearly identifies the specific enzyme isoform. A widely accepted nomenclature (3), which will be used in these Perspective articles, identifies the three mammalian enzyme isoforms as nNOS, iNOS, and eNOS, reflecting the tissues of origin for the original protein and cDNA isolates. As denoted by its prefix, nNOS was originally purified and cloned from neuronal tissues. However, nNOS is now known to be much more widely distributed, with an important level of expression in skeletal muscle. iNOS, originally purified and cloned from an immunoactivated macrophage cell line, has since been identified in myriad mammalian tissues, and iNOS expression has been studied in cells as diverse as cardiac myo- cytes, glial cells, and vascular smooth muscle cells (to name only a few). eNOS, the last of the three mammalian NOS iso- forms to be isolated, was originally purified and cloned from vascular endothelium, but has since been discovered in cardiac myocytes, blood platelets, brain (hippocampus), and else- where. To add to the confusion, the human genes for the NOS isoforms are officially categorized in the order of their isola- tion and characterization; thus, the human genes encoding nNOS, iNOS, and eNOS are termed NOS1 , NOS2 , and NOS3 , respectively.
Whether nitric oxide (NO) is involved in cutaneous active vasodilation during hyperthermia in humans is unclear. We tested for a role of NO in this process during heat stress (water-perfused suits) in seven healthy subjects. Two forearm sites were instrumented with intradermal microdialysis probes. One site was perfused with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) dissolved in Ringer solution to abolish NO production. The other site was perfused with Ringer solution only. At those sites, skin blood flow (laser-Doppler flowmetry) and sweat rate were simultaneously and continuously monitored. Cutaneous vascular conductance, calculated from laser-Doppler flowmetry and mean arterial pressure, was normalized to maximal levels as achieved by perfusion with the NO donor nitroprusside through the microdialysis probes. Under normothermic conditions, L-NAME did not significantly reduce cutaneous vascular conductance. During hyperthermia, with skin temperature held at 38-38.5 degreesC, internal temperature rose from 36.66 +/- 0.10 to 37.34 +/- 0.06 degreesC (P < 0.01). Cutaneous vascular conductance at untreated sites increased from 12 +/- 2 to 44 +/- 5% of maximum, but only rose from 13 +/- 2 to 30 +/- 5% of maximum at L-NAME-treated sites (P < 0.05 between sites) during heat stress. L-NAME had no effect on sweat rate (P > 0.05). Thus cutaneous active vasodilation requires functional NO synthase to achieve full expression.
Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.
Endothelial nitric oxide (NO) synthase (eNOS) is regulated by heat shock protein 90 (HSP90), a heat-inducible protein; however, the effect of heat shock on eNOS expression and eNO release is unknown. Bovine aortic endothelial cells were incubated for 1 h at 37 degrees C, 42 degrees C, or 45 degrees C and cell lysates were evaluated with the use of Western blotting. We observed a 2.1 +/- 0.1-fold increase in eNOS protein content, but no change in HSP90 content, HSP70 content, or HSP90/eNOS association, 24 h after heat shock at 42 degrees C. We also observed a 7.7 +/- 1.5-fold increase in HSP70 protein content, but did not observe a change in eNOS or HSP90 24 h after heat shock at 45 degrees C. eNOS activity and maximal bradykinin-stimulated NO release was significantly increased 24 h after heat shock at 42 degrees C. Heat shock in rats (core temperature: 42 degrees C, 15 min) resulted in a significant increase in aortic eNOS, HSP90, and HSP70 protein content. The aorta from heat-shocked rats exhibited a decreased maximal contractile response to phenylephrine, which was abolished by preincubation with NG-nitro-l-arginine. We conclude that prior heat shock is a physical stimulus of increased eNOS expression and is associated with an increase in eNOS activity, agonist-stimulated NO release, and a decreased vasoconstrictor response.
Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-l-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G(2)/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses.
Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.
Sustained sympathetic activation not only leads to vasoconstriction but also might induce paradox vasodilation. This study was performed to explore whether and how alpha(2)-receptor stimulation mediates this vasodilation. We investigated 11 healthy subjects in 33 dermal microdialysis (MD) sessions. After nerve trunk blockade, MD fibers were inserted and perfused with physiological saline until skin trauma-related vasodilation subsided. Thereafter, fibers were perfused with either clonidine solutions (10(-3), 5 x 10(-4), 10(-4) mol/l), N(G)-monomethyl-l-arginine (L-NMMA; nitric oxide synthase blocker), acetylsalicylic acid (ASA; cyclooxygenase blocker), or combinations of these. Laser-Doppler scanning of the investigated skin revealed that clonidine not only induces vasoconstriction but subsequently also vasodilation with higher concentrations (P < 0.001). In contrast, both L-NMMA and ASA induced vasoconstriction (P < 0.001). By coapplication of 10(-3) mol/l clonidine with L-NMMA or ASA, vasodilation was partially prevented (P < 0.001). Our results demonstrate that sustained alpha(2)-receptor stimulation induces vasodilation in a dose-dependent way, which is mediated by nitric oxide and prostaglandin mechanisms in human skin.
Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.
To assess endothelin-1 (ET-1) response to cold stimulation in essential acrocyanosis (EA), the authors measured ET-1 plasma concentrations in 6 patients with EA (6 women, age range seventeen to thirty-seven years) and in 6 controls (5 women, 1 man, age range twenty-one to thirty-seven years) before and after cold challenge by unilateral hand immersion in water bath at 13°C for five minutes. The contralateral upper limb was considered as control. Blood samples were simultaneously drawn from an antecubital vein in the cooled side and in the contralateral upper limb at baseline, at the end of cooling, and at ten and ninety minutes after cooling was begun. Plasma ET-1 was deter mined by a radioimmunoassay system. Results are mean ±SD. Baseline ET-1 was higher in patients with EA (5.1 ±0.3 pmol/L) than in controls (1.9 ± 0.1 pmol/L, P < 0.001). After hand cooling, ET-1 in the cold-exposed upper limb rose in patients with EA to a peak value of 7.2 ±0.7 pmol/L, which was greater than that observed in healthy subjects (2.7 ±0.4 pmol/L, P < 0.001). The absolute increase in ET-1 plasma concentrations from baseline to peak value was significantly higher in patients with EA than in controls (2.1 ±0.3 vs 0.8 ± 0.2 pmol/L, respectively, P < 0.001). In patients with EA, but not in controls, the rise in ET-1 plasma concentrations was still detected ninety minutes after cooling. The same time course of the plasma ET-1 concentrations was observed in the noncooled upper limb, but the increases in ET-1 at different times after cold stimulus were smaller than in the cold-challenged upper limb in both groups (P < 0.001). In conclusion, the results demonstrate that in patients with EA, baseline plasma levels of ET-1 are enhanced and are further increased by cooling until ninety minutes after cold challenge. This rise in plasma ET-1 could contribute to potentiating and prolonging cold-induced vasoconstric tion/vasospasm and/or could be a marker for endothelial damage in EA.
Endothelial cell functions, such as arachidonic acid metabolism, may be modulated by membrane stresses induced by blood flow.
The production of prostacyclin by primary human endothelial cell cultures subjected to pulsatile and steady flow shear stress
was measured. The onset of flow led to a sudden increase in prostacyclin production, which decreased to a steady rate within
several minutes. The steady-state production rate of cells subjected to pulsatile shear stress was more than twice that of
cells exposed to steady shear stress and 16 times greater than that of cells in stationary culture.
The role of adrenergic nerve function in the cutaneous vascular response to changes in local skin temperature in the human forearm was examined using three protocols: 1) blocking release of norepinephrine presynaptically by local iontophoresis of bretylium (BT), 2) altering background adrenergic tone by changing whole body skin temperature, and 3) blocking cutaneous nerves by proximal infiltration of local anesthetic. Forearm skin blood flow was measured by laser-Doppler flowmetry (LDF) and cutaneous vascular conductance (CVC) was calculated as LDF/blood pressure. In protocol 1, local cooling (29 degrees C) elicited a rapid and sustained fall in CVC at control sites (-43 +/- 8%) in contrast to a biphasic response at BT-treated sites, consisting of an initial vasodilation followed by a vasoconstriction (percent change CVC = 28 +/- 13 and -34 +/- 18, respectively). Local warming (39 degrees C) increased CVC at control and at BT-treated sites by 331 +/- 46 and 139 +/- 31%, respectively. In protocol 2, at a neutral, cool, or warm whole body skin temperature, local cooling (29 degrees C) elicited similar reductions in CVC (-34 +/- 8, -29 +/- 5, and -30 +/- 4%, respectively), and local warming (38 degrees C) produced similar increases in CVC (89 +/- 15, 85 +/- 21, and 74 +/- 22%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca2+]i). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar flow chamber or treated with Ca2+ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm2) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca2+]i caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca2+ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca2+ requirement for flow-dependent NO production, we measured [Ca2+]i with a novel flow-step protocol. [Ca2+]i increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca2+]i, and they suggest that phosphorylation of ecNOS may importantly modulate its activity during the imposition of increased shear stress.
Mechanical forces are important modulators of cellular function in many tissues and are particularly important in the cardiovascular system. The endothelium, by virtue of its unique location in the vessel wall, responds rapidly and sensitively to the mechanical conditions created by blood flow and the cardiac cycle. In this study, we examine data which suggest that steady laminar shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. We explore the ability of shear stress to modulate atherogenesis via its effects on endothelial-mediated alterations in coagulation, leukocyte and monocyte migration, smooth muscle growth, lipoprotein uptake and metabolism, and endothelial cell survival. We also propose a model of signal transduction for the endothelial cell response to shear stress including possible mechanotransducers (integrins, caveolae, ion channels, and G proteins), intermediate signaling molecules (c-Src, ras, Raf, protein kinase C) and the mitogen activated protein kinases (ERK1/2, JNK, p38, BMK-1), and effector molecules (nitric oxide). The endothelial cell response to shear stress may also provide a mechanism by which risk factors such as hypertension, diabetes, hypercholesterolemia, and sedentary lifestyle act to promote atherosclerosis.
Local warming of skin induces vasodilation by unknown mechanisms. To test whether nitric oxide (NO) is involved, we examined effects of NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on vasodilation induced by local warming of skin in six subjects. Two adjacent sites on the forearm were instrumented with intradermal microdialysis probes for delivery of L-NAME and sodium nitroprusside. Skin blood flow was monitored by laser-Doppler flowmetry (LDF) at microdialysis sites. Local temperature (Tloc) of the skin at both sites was controlled with special LDF probe holders. Mean arterial pressure (MAP; Finapres) was measured and cutaneous vascular conductance calculated (CVC = LDF/MAP = mV/mmHg). Data collection began with a control period (Tloc at both sites = 34 degrees C). One site was then warmed to 41 degrees C while the second was maintained at 34 degrees C. Local warming increased CVC from 1.44 +/- 0.41 to 4.28 +/- 0.60 mV/mmHg (P < 0.05). Subsequent L-NAME administration reduced CVC to 2.28 +/- 0.47 mV/mmHg (P < 0.05 vs. heating), despite the continued elevation of Tloc. At a Tloc of 34 degrees C, L-NAME reduced CVC from 1.17 +/- 0.23 to 0.75 +/- 0.11 mV/mmHg (P < 0.05). Administration of sodium nitroprusside increased CVC to levels no different from those induced by local warming. Thus NOS inhibition attenuated, and sodium nitroprusside restored, the cutaneous vasodilation induced by elevation of Tloc; therefore, the mechanism of cutaneous vasodilation by local warming requires NOS generation of NO.
Cold-induced vasodilation (CIVD) in the finger tips generally occurs 5-10 min after the start of local cold exposure of the extremities. This phenomenon is believed to reduce the risk of local cold injuries. However, CIVD is almost absent during hypothermia, when survival of the organism takes precedence over the survival of peripheral tissue. Subjects that are often exposed to local cold (e.g. fish filleters) develop an enhanced CIVD response. Also, differences between ethnic groups are obvious, with black people having the weakest CIVD response. Many other factors affect CIVD, such as diet, alcohol consumption, altitude, age and stress. CIVD is probably caused by a sudden decrease in the release of neurotransmitters from the sympathetic nerves to the muscular coat of the arterio-venous anastomoses (AVAs) due to local cold. AVAs are specific thermoregulatory organs that regulate blood flow in the cold and heat. Their relatively large diameter enables large amounts of blood to pass and convey heat to the surrounding tissue. Unfortunately, information on the quantity of AVAs is lacking, which makes it difficult to estimate the full impact on peripheral blood flow. This review illustrates the thermospecificity of the AVAs and the close link to CIVD. CIVD is influenced by many parameters, but controlled experiments yield information on how CIVD protects the extremities against cold injuries.
Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.
Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.
Previous work indicates that sympathetic nerves participate in the vascular responses to direct cooling of the skin in humans. We evaluated this hypothesis further in a four-part series by measuring changes in cutaneous vascular conductance (CVC) from forearm skin locally cooled from 34 to 29 degrees C for 30 min. In part 1, bretylium tosylate reversed the initial vasoconstriction (-14 +/- 6.6% control CVC, first 5 min) to one of vasodilation (+19.7 +/- 7.7%) but did not affect the response at 30 min (-30.6 +/- 9% control, -38.9 +/- 6.9% bretylium; both P < 0.05, P > 0.05 between treatments). In part 2, yohimbine and propranolol (YP) also reversed the initial vasoconstriction (-14.3 +/- 4.2% control) to vasodilation (+26.3 +/- 12.1% YP), without a significant effect on the 30-min response (-26.7 +/- 6.1% YP, -43.2 +/- 6.5% control; both P < 0.05, P > 0.05 between sites). In part 3, the NPY Y1 receptor antagonist BIBP 3226 had no significant effect on either phase of vasoconstriction (P > 0.05 between sites both times). In part 4, sensory nerve blockade by anesthetic cream (Emla) also reversed the initial vasoconstriction (-20.1 +/- 6.4% control) to one of vasodilation (+213.4 +/- 87.0% Emla), whereas the final levels did not differ significantly (-37.7 +/- 10.1% control, -37.2 +/- 8.7% Emla; both P < 0.05, P > 0.05 between treatments). These results indicate that local cooling causes cold-sensitive afferents to activate sympathetic nerves to release norepinephrine, leading to a local cutaneous vasoconstriction that masks a nonneurogenic vasodilation. Later, a vasoconstriction develops with or without functional sensory or sympathetic nerves.
Cold-induced vasodilation (CIVD) is a cyclic oscillation in blood flow that occurs in the extremities on cold exposure and that is likely associated with reduced risk of cold injury (e.g., frostbite) as well as improved manual dexterity and less pain while working in the cold. The CIVD response varies between individuals, but the within-subject reproducibility has not been adequately described. The purpose of this study was to quantify the within-subject variability in the CIVD response under standardized conditions. Twenty-one volunteers resting in a controlled environment (27 degrees C) immersed the middle finger in warm water (42 degrees C) for 15 min to standardize initial finger temperature and then in cold water (4 degrees C; CWI) for 30 min, on five separate occasions. Skin temperature (Tf) and blood flow (laser-Doppler; expressed as percent change from warm-water peak) responses that describe CIVD were identified, including initial nadir reached during CWI, onset time of CIVD, initial apex during CIVD, time of that apex, and overall mean during CWI. Within-subject coefficient of variation for Tf across the five tests for the nail bed and pad, respectively, were as follows: nadir, 9 and 21%; onset, 18 and 19%; apex, 12 and 17%; apex time, 23 and 24%; mean 10 and 15%. For blood flow, these values were as follows: nadir 52 and 64%; onset, 6 and 5%; apex, 33 and 31%; apex time 9 and 8%; and mean 43 and 34%. Greater variability was found in the temperature response of the finger pad than the nail bed, but for blood flow the variability was similar between locations. Variability in onset and apex time between sites was similar for both temperature and blood flow responses. The reproducibility of the time course of CIVD suggests this methodology may be of value for further studies examining the mechanism of the response.
This review focuses on the neural and local mechanisms that have been demonstrated to effect cutaneous vasodilation and vasoconstriction in response to heat and cold stress in vivo in humans. First, our present understanding of the mechanisms by which sympathetic cholinergic nerves mediate cutaneous active vasodilation during reflex responses to whole body heating is discussed. These mechanisms include roles for cotransmission as well as nitric oxide (NO). Next, the mechanisms by which sympathetic noradrenergic nerves mediate cutaneous active vasoconstriction during whole body cooling are reviewed, including cotransmission by neuropeptide Y (NPY) acting through NPY Y1 receptors. Subsequently, current concepts for the mechanisms that effect local cutaneous vascular responses to direct skin warming are examined. These mechanisms include the roles of temperature-sensitive afferent neurons as well as NO in causing vasodilation during local heating of skin. This section is followed by a review of the mechanisms that cause local cutaneous vasoconstriction in response to direct cooling of the skin, including the dependence of these responses on intact sensory and sympathetic, noradrenergic innervation as well as roles for nonneural mechanisms. Finally, unresolved issues that warrant further research on mechanisms that control cutaneous vascular responses to heating and cooling are discussed.
Vascular endothelium–derived nitric oxide (NO), originally identified as endothelium-derived relaxing factor,1–3 plays a pivotal role in regulation of vascular homeostasis.4 NO is a major regulator of vascular tone and blood pressure, and has multiple antiatherogenic roles including antiinflammatory, antithrombotic, antiproliferative, and antioxidant effects.4–6 Loss of the bioavailability of endothelium-derived NO is the hallmark of endothelial dysfunction and is implicated in the pathogenesis of cardiovascular disease such as hypertension and atherosclerosis.7,8 Therefore, it is of great interest to understand the molecular mechanisms regulating NO production by endothelium, which is likely to provide new insight into endothelial function in health and disease.
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Endothelial NO synthase (eNOS) is a highly regulated, calcium (Ca2+)/calmodulin (CaM)-dependent enzyme responsible for the physiological production of NO in the vasculature.9,10 In endothelial cells (ECs), NO is formed from its precursor L-arginine via the enzymatic activation of eNOS with cofactors such as tetrahydrobiopterin (BH4). eNOS activation and subsequent NO production is stimulated by a variety of physical stimuli such as fluid shear stress generated by blood flow and by many humoral factors including acetylcholine, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin.10–12 Increasing evidence suggest that eNOS is regulated by subcellular localization,9,13,14 posttranslational modifications such as phosphorylation at serine 1179 (S1179) by Akt,15–17 and interactions with several regulatory proteins such as heat shock protein 90 (HSP90) and caveolin-1.18,19 In particular, subcellular localization of eNOS is critical for optimal coupling of extracellular stimulation to NO production.10 In ECs, eNOS appears to localize at peripheral aspects of the Golgi complex and cholesterol-rich microdomains of the plasma membrane (PM) such as caveolae. Cotranslational N-myristoylation and posttranslational cysteine palmitoylation of eNOS determine its membrane targeting.9,13 It has been proposed that eNOS membrane localization may bring eNOS …