Reactive Oxygen Species Are Essential for Vasoconstriction upon Cold Exposure

Purpose. We explored the role of ROS in cold-induced vasoconstriction and corresponding mechanism. Methods. Three experiments were performed. First, we measured blood flow in human hands before and after cold exposure. Second, 24 mice were randomly divided into 3 groups: 8 mice received saline injection, 8 received subcutaneous Tempol injection, and 8 received intrathecal Tempol injection. After 30 min, we determined blood flow in the skin before and after cold exposure. Finally, we used Tempol, CCG-1423, and Go 6983 to pretreat HAVSMCs and HUVECs for 24 h. Then, cells in the corresponding groups were exposed to cold (6 h, 4°C). After cold exposure, the cytoskeleton was stained. Intracellular Ca2+ and ROS levels were measured by flow cytometry and fluorescence microscopy. We measured protein expression via Western blotting. Results. In the first experiment, after cold exposure, maximum skin blood flow decreased to 118.4 ± 50.97 flux units. Then, Tempol or normal saline pretreatment did not change skin blood flow. Unlike intrathecal Tempol injection, subcutaneous Tempol injection increased skin blood flow after cold exposure. Finally, cold exposure for 6 h shrank the cells, making them narrower, and increased intracellular Ca2+ and ROS levels in HUVECs and HAVSMCs. Tempol reduced cell shrinkage and decreased intracellular Ca2+ levels. In addition, Tempol decreased intracellular ROS levels. Cold exposure increased RhoA, Rock1, p-MLC-2, ET-1, iNOS, and p-PKC expression and decreased eNOS expression. Tempol or CCG-1423 pretreatment decreased RhoA, Rock1, and p-MLC-2 levels in HAVSMCs. Furthermore, Tempol or Go 6983 pretreatment decreased ET-1, iNOS, and p-PKC expression and increased eNOS expression in HUVECs. Conclusion. ROS mediate the vasoconstrictor response within the cold-induced vascular response, and ROS in blood vessel tissues rather than nerve fibers are involved in vasoconstriction via the ROS/RhoA/ROCK1 and ROS/PKC/ET-1 pathways in VSMCs and endothelial cells.

Enhanced Vasoconstriction Mediated by α1-Adrenergic Mechanisms in Small Femoral Arteries in Newborn Llama and Sheep Gestated at Low and High Altitudes

The authors previously demonstrated that newborn llama (NBLL) express high levels of α1 adrenergic receptors, which provide a potent vasoconstriction response when compared with newborn sheep (NBSH) gestated at sea level. However, data regarding the impact of chronic gestational hypobaric hypoxia on α-adrenergic vasoconstriction in the neonatal life has not been studied. We evaluated if gestation under chronic hypobaric hypoxia modifies α1-adrenergic vasoconstrictor function in NBLL and NBSH. We compared the vasoconstrictor response induced by potassium and α-adrenergic stimuli in isolated small femoral arteries of NBLL and NBSH gestated at high altitude (HA; 3,600 m) or low altitude (LA; 580 m). The maximal contraction (RMAX) and potency (EC50) to potassium, noradrenaline (NA), and phenylephrine (PHE) were larger in HA-NBLL than LA-NBLL. RMAX to potassium, NA, and PHE were lower in HA-NBSH when compared with LA-NBSH and potency results were similar. Competitive blockade with prazosin showed that RNLL LA/HA have a similar pA2. In contrast, NBSH had increased pA2 values in HA when compared with LA. Finally, small femoral arteries denudated or treated with LNAME in LA and HA lacked NO or endothelium participation in response to PHE stimulation. In contrast, NBSH displayed that denudation or blockade with LNAME support NO or endothelium participation in response to PHE activation. In conclusion, HA chronic hypoxia enhances α1 adrenergic receptor activity in small femoral arteries in NBLL to a higher degree than NBSH, implying a higher vasoconstriction function.

Long Term Cerebrovascular Dysfunction in the Offspring from Maternal Electronic Cigarette Use during Pregnancy

Electronic cigarettes (E-cigs) have been promoted as harm-free or less-risky than smoking, even for women during pregnancy. These claims are made largely on E-cig aerosol having fewer number of toxic chemicals compared to cigarette smoke. Given that even low levels of smoking are found to produce adverse birth outcomes, we sought to test the hypothesis that vaping during pregnancy (with or without nicotine) would not be harm-free, and would result in vascular dysfunction that would be evident in offspring during adolescent and/or adult life. Pregnant female Sprague Dawley rats were exposed to E-cig aerosol (1-hour/day, 5 days/week, starting on gestational day 2 until pups were weaned) using e-liquid with 0 mg/ml (E-cig0) or 18 mg/ml nicotine (E-cig18) and compared to ambient air exposed controls. Body mass at birth and at weaning were not different between groups. Assessment of middle cerebral artery (MCA) reactivity revealed a 51-56% reduction in endothelial-dependent dilation response to acetylcholine (ACh) for both E-cig0 and E-cig18 in 1-month, 3-month (adolescent), and 7-month old (adult) offspring (p<0.05 compared to air, all time points). MCA response to sodium nitroprusside (SNP) and myogenic tone were not different across groups suggesting that endothelial-independent responses were not altered. The MCA vasoconstrictor response (5-hydroxytryptamine, 5-HT) was also not different across treatment and age groups. These data demonstrate that maternal vaping during pregnancy is not harm-free and confers significant cerebrovascular health risk/dysfunction to offspring that persists into adult life.

ATP and acetylcholine interact to modulate vascular tone and α1-adrenergic vasoconstriction in humans

The vascular endothelium senses and integrates numerous inputs to regulate vascular tone. Recent evidence reveals complex signal processing within the endothelium, yet little is known about how endothelium-dependent stimuli interact to regulate blood flow. We tested the hypothesis that combined stimulation of the endothelium with adenosine triphosphate (ATP) and acetylcholine (ACh) elicits greater vasodilation and attenuates α1‑adrenergic vasoconstriction compared to combination of ATP or ACh with the endothelium-independent dilator sodium nitroprusside (SNP). We assessed forearm vascular conductance (FVC) in young adults (6F, 7M) during local intra-arterial infusion of ATP, ACh, or SNP alone and in the following combinations: ATP+ACh, SNP+ACh, and ATP+SNP wherein the second dilator was co-infused after attaining steady-state with the first dilator. By design, each dilator evoked a similar response when infused separately (ΔFVC, ATP: 48±4; ACh: 57±6; SNP: 53±6 ml·min-1·100 mmHg-1; P≥0.62). Combined infusion of the endothelium-dependent dilators evoked greater vasodilation than combination of either dilator with SNP (ΔFVC from first dilator, ATP+ACh: 45±9 vs. SNP+ACh: 18±7 and ATP+SNP: 26±4 ml·min-1·100 mmHg-1, P<0.05). Phenylephrine was subsequently infused to evaluate α1‑adrenergic vasoconstriction. Phenylephrine elicited less vasoconstriction during infusion of ATP or ACh vs. SNP (ΔFVC, -25±3 and -29±4 vs. -48±3%; P<0.05). The vasoconstrictor response to phenylephrine was further diminished during combined infusion of ATP+ACh (-13±3%; P<0.05 vs. ATP or ACh alone) and was less than that observed when either dilator was combined with SNP (SNP+ACh: -26±3%; ATP+SNP: -31±4%; both P<0.05 vs. ATP+ACh). We conclude that endothelium-dependent agonists interact to elicit vasodilation and limit α1‑adrenergic vasoconstriction in humans.

A Blunted Sympathetic Function and an Enhanced Nitrergic Activity Contribute to Reduce Mesenteric Resistance in Hyperthyroidism

We aimed to determine whether an experimental model of hyperthyroidism could alter the function of sympathetic and nitrergic components of mesenteric innervation. For this purpose, male Wistar rats were divided into (1) control rats (CT) and (2) rats infused with L-Thyroxine (HT). Body weight gain and adipose tissue accumulation were lower in HT rats, while systolic blood pressure and citrate synthase activity in the soleus muscle were increased by HT. In segments from the superior mesenteric artery, the application of an electrical field stimulation (EFS) induced a vasoconstrictor response, which was lower in arteries from HT animals. The alpha-adrenoceptor antagonist phentolamine diminished EFS-induced vasoconstriction to a lower extent in HT arteries, while the purinergic receptor antagonist suramin reduced contractile response to EFS only in segments from CT. In line with this, noradrenaline release, tyrosine hydroxylase expression and activation and dopamine β hydroxylase expression were diminished in HT. The unspecific nitric oxide synthase (NOS) inhibitor L-NAME increased EFS-induced vasoconstriction more markedly in segments from HT rats. NO release was enhanced in HT, probably due to an enhancement in neuronal NOS activity, in which a hyperactivation of both PKC and PI3K-AKT signaling pathways might play a relevant role. In conclusion, perivascular mesenteric innervation might contribute to reduce the vascular resistance observed in hyperthyroidism.

Endothelin receptor heteromerization inhibits β-arrestin function in HEK293 cells

The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca2+ transients, leading to prolongation of Gαq-dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to β-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in β-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced β-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq-dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq-dependent signaling and inhibition of β-arrestin function.

Aortae of Young Rats with Metabolic Syndrome Exhibit Enhanced Vasoconstrictor Activity Than Older Rats

Background Metabolic syndrome (MetS) causes pathological remodeling of the heart and adjacent vessels. The functional changes in the big vessels in different age groups had not been fully delineated. Aim of the work The present study was planned to investigate aortic vasodilator and vasoconstrictor reactivity in young, adult and old female rats with MetS. Design: The experimental study was performed on 90 female albino rats randomized into 6 groups: young, adult and old rats with MetS and their respective control groups. Methods MetS was induced by feeding rats 41% fructose -containing diet and giving fructose solution (5 g fructose in 4 ml distilled water/day) by gavage in two sessions (2 ml/session). On the 8th week, all rats were sacrificed and were subjected to determination of body weight (BW), body mass index (BMI), absolute and relative visceral fat weight (VF), fasting blood glucose (FBG), plasma insulin (PI), homeostasis model of insulin resistance (HOMA-IR) and plasma lipid profile. All rats’ aortae were subjected to study of vascular reactivity to Potassium chloride (KCL), phenylephrine (PE) and acetyl choline (A.Ch) as well as estimation of nitrite content. Results On the 8th week of the study, all MetS groups developed criteria of metabolic syndrome as evidenced by the significant increase in final BW, BMI, absolute and relative VF weights, FBG, PI and HOMA-IR compared to their control group values. Also, MetS rat groups exhibited evident dyslipidemia in the form of significant increase in plasma levels of triglycerides, total cholesterol and significant decrease in HDL-cholesterol compared to their control group values. Aortae of young and adult MetS rat groups showed significant increase in their vasoconstrictor response to KCl and PE and decrease in A.Ch/KCL% and A.Ch/PE % compared to their controls, while old MetS rat group showed significantly increased vasoconstrictor response only to KCL compared to their controls. When compared to each other, young age MetS group had significantly higher vasoconstrictor response to PE compared to old MetS group despite comparable nitrite content. Conclusion Met.S causes functional vascular changes in all age groups with unexpectedly enhanced vasoconstrictor response in the young group compared to old.