Limits of Ultra: Towards an Interdisciplinary Understanding of Ultra-Endurance Running Performance.

Ultra-endurance running (UER) poses extreme mental and physical challenges that present many barriers to completion, let alone performance. Despite these challenges, participation in UER events continues to increase. With the relative paucity of research into UER training and racing compared with traditional endurance running distance (e.g., marathon), it follows that there are sizable improvements still to be made in UER if the limitations of the sport are sufficiently understood. The purpose of this review is to summarise our current understanding of the major limitations in UER. We begin with an evolutionary perspective that provides the critical background for understanding how our capacities, abilities and limitations have come to be. Although we show that humans display evolutionary adaptations that may bestow an advantage for covering large distances on a daily basis, these often far exceed the levels of our ancestors, which exposes relative limitations. From that framework, we explore the physiological and psychological systems required for running UER events. In each system, the factors that limit performance are highlighted and some guidance for practitioners and future research are shared. Examined systems include thermoregulation, oxygen delivery and utilisation, running economy and biomechanics, fatigue, the digestive system, nutritional and psychological strategies. We show that minimising the cost of running, damage to lower limb tissue and muscle fatigability may become crucial in UER events. Maintaining a sustainable core body temperature is critical to performance, and an even pacing strategy, strategic heat acclimation and individually calculated hydration all contribute to sustained performance. Gastrointestinal issues affect almost every UER participant and can be due to a variety of factors. We present nutritional strategies for different event lengths and types, such as personalised and evidence-based approaches for varying types of carbohydrate, protein and fat intake in fluid or solid form, and how to avoid flavour fatigue. Psychology plays a vital role in UER performance, and we highlight the need to be able to cope with complex situations, and that specific long and short-term goal setting improves performance. Fatigue in UER is multi-factorial, both physical and mental, and the perceived effort or level of fatigue have a major impact on the ability to continue at a given pace. Understanding the complex interplay of these limitations will help prepare UER competitors for the different scenarios they are likely to face. Therefore, this review takes an interdisciplinary approach to synthesising and illuminating limitations in UER performance to assist practitioners and scientists in making informed decisions in practice and applicable research.

Pericytes improve locomotor recovery after spinal cord injury in male and female neonatal rats.

OBJECTIVE: It is not known how activation of the hypoxia-inducible factor (HIF) pathway in pericytes, cells of the microvascular wall, influences new capillary growth. We tested the hypothesis that HIF-activated pericytes promote angiogenesis in a neonatal model of spinal cord injury (SCI). METHODS: Human placental pericytes stimulated with cobalt chloride and naïve pericytes were injected into the site of a thoracic hemi-section of the spinal cord in rat pups on postnatal day three (P3). Hindlimb motor recovery and Doppler blood flow perfusion at the site of transection were measured on P10. Immunohistochemistry was used to visualize vessel and neurofilament density for quantification. RESULTS: Injection of HIF-activated pericytes resulted in greater vascular density in males but did not result in improved motor function for males or females. Injection of non-HIF-activated pericytes resulted improved motor function recovery in both sexes (males, 2.722 ± 0.31-fold score improvement; females, 3.824 ± 0.58-fold score improvement, P < .05) but produced no significant changes in vessel density. CONCLUSIONS: HIF-activated pericytes promote vascular density in males post-SCI. Acute delivery of non-HIF-activated pericytes at the site of injury can improve motor recovery post-SCI.

Maternal exposure to carbamazepine at environmental concentrations can cross intestinal and placental barriers.

Psychoactive pharmaceuticals have been found as teratogens at clinical dosage during pregnancy. These pharmaceuticals have also been detected in minute (ppb) concentrations in drinking water in the US, and are environmental contaminants that may be complicit in triggering neurological disorders in genetically susceptible individuals. Previous studies have determined that psychoactive pharmaceuticals (fluoxetine, venlafaxine and carbamazepine) at environmentally relevant concentrations enriched sets of genes regulating development and function of the nervous system in fathead minnows. Altered gene sets were also associated with potential neurological disorders, including autism spectrum disorders (ASD). Subsequent in vitro studies indicated that psychoactive pharmaceuticals altered ASD-associated synaptic protein expression and gene expression in human neuronal cells. However, it is unknown if environmentally relevant concentrations of these pharmaceuticals are able to cross biological barriers from mother to fetus, thus potentially posing risks to nervous system development. The main objective of this study was to test whether psychoactive pharmaceuticals (fluoxetine, venlafaxine, and carbamazepine) administered through the drinking water at environmental concentrations to pregnant mice could reach the brain of the developing embryo by crossing intestinal and placental barriers. We addressed this question by adding (2)H-isotope labeled pharmaceuticals to the drinking water of female mice for 20 days (10 pre-and 10 post-conception days), and quantifying (2)H-isotope enrichment signals in the dam liver and brain of developing embryos using isotope ratio mass spectrometry. Significant levels of (2)H enrichment was detected in the brain of embryos and livers of carbamazepine-treated mice but not in those of control dams, or for fluoxetine or venlafaxine application. These results provide the first evidence that carbamazepine in drinking water and at typical environmental concentrations is transmitted from mother to embryo. Our results, combined with previous evidence that carbamazepine may be associated with ASD in infants, warrant the closer examination of psychoactive pharmaceuticals in drinking water and their potential association with neurodevelopmental disorders.

Driving the Hypoxia-Inducible Pathway in Human Pericytes Promotes Vascular Density in an Exosome-Dependent Manner.

OBJECTIVES: The mechanisms involved in activating pericytes, cells that ensheath capillaries, to engage in the formation of new capillaries, angiogenesis, remain unknown. In this study, the hypothesis was tested that pericytes could be stimulated to promote angiogenesis by driving the HIF pathway. METHODS: Pericytes were stimulated with CoCl2 to activate the HIF pathway. Stimulated pericytes were cocultured with endothelial cells in a wound healing assay and in a 3D collagen matrix assay of angiogenesis. A culture system of spinal cord tissue was used to assess microvascular outcomes after treatment with stimulated pericytes. Pharmaceutical inhibition of exosome production was also performed. RESULTS: Treatment with stimulated pericytes resulted in faster wound healing (1.92 ± 0.18 fold increase, p < 0.05), greater endothelial cord formation (2.9 ± 0.14 fold increase, p < 0.05) in cell culture assays, and greater vascular density (1.78 ± 0.23 fold increase, p < 0.05) in spinal cord tissue. Exosome secretion and the physical presence of stimulated pericytes were necessary in the promotion of angiogenic outcomes. CONCLUSIONS: These results elucidate a mechanism that may be exploited to enhance features of angiogenesis in the CNS.

The connexin 43/ZO-1 complex regulates cerebral endothelial F-actin architecture and migration.

Endothelial cell migration is a fundamental process during angiogenesis and, therefore, a point of intervention for therapeutic strategies aimed at controlling pathologies involving blood vessel growth. We sought to determine the role of the gap junction protein connexin 43 (Cx43) in key features of angiogenesis in the central nervous system. We used an in vitro model to test the hypothesis that a complex of interacting proteins, including Cx43 and zonula occludens-1 (ZO-1), regulates the migratory behavior of cerebral endothelium. With knockdown and overexpression experiments, we demonstrate that the rate of healing following scrape-wounding of endothelium is regulated by the level of Cx43 protein expression. The effects on cell motility and proliferation were independent of gap junction communication as cells were sensitive to altered Cx43 expression in single plated cells. Coupling of Cx43/ZO-1 critically regulates this process as demonstrated with the use of a Cx43 α-carboxy terminus 1 peptide mimetic (αCT1) and overexpression of a mutant ZO-1 with the Cx43-binding PDZ2 domain deleted. Disrupting the Cx43/ZO-1 complex with these treatments resulted in collapse of the organized F-actin cytoskeleton and the appearance of actin nodes. Preincubation with the myosin 2 inhibitors blebbistatin or Y-27632 disrupted the Cx43/ZO-1 complex and inhibited cell spreading at the leading edge of migration. Cells studied individually in time-lapse open field locomotion assays wandered less when Cx43/ZO-1 interaction was disrupted without significant change in speed, suggesting that faster wound healing is a product of linearized migration. In contrast to the breakdown of F-actin architecture, microtubule architecture was not obviously affected by treatments. This study provides new insight into the fundamental regulatory mechanisms of cerebral endothelial cell locomotion. Cx43 tethers the F-actin cytoskeleton through a ZO-1 linker and supports cell spreading and exploration during locomotion. Here, we demonstrate that releasing this actin-coupled tether shifts the balance of directional migration control to a more linear movement that enhances the rate of wound healing.

Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.

We previously identified and characterized a 66-68 kDa membrane-associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX)-binding protein. In order to further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells, we first showed that HSC70 colocalizes and interacts with reduced folate carrier (RFC) in L1210 cells by confocal laser scanning microscopy and Duolink in situ proximity ligation assay. The tyrosine phosphorylation status of HSC70 found in the membrane fraction was different from the parental L1210/0 and cisplatin (CDDP)-MTX cross resistant L1210/DDP cells. In MTX-binding assays, HSC70 from L1210/DDP cells showed less affinity for MTX-agarose beads than that of L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. Moreover, site-directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These findings suggest that tyrosine phosphorylation of HSC70 regulates the transportation of MTX into the cells via the HSC70-RFC system and contributes to MTX resistance in L1210 cells.

Alterations of retinal vasculature in cystathionine-ß-synthase heterozygous mice: a model of mild to moderate hyperhomocysteinemia.

Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-ß-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.

Homocysteine disrupts outgrowth of microvascular endothelium by an iNOS-dependent mechanism.

OBJECTIVE: To test the hypothesis that Hcy impairs angiogenic outgrowth through an iNOS-dependent mechanism. METHODS: Adult C57Bl/6 mouse choroid explants were used in angiogenic outgrowth assays. Mouse microvascular endothelial cells were studied in culture during scrape-induced migration and dispersed cell locomotion experiments. Activity of iNOS was manipulated with pharmacology (1400 W), siRNA, and by use of choroid explants from iNOS knockout mice (iNOS(-/-)). RESULTS: Hcy (20 µM) significantly decreased the area of endothelial outgrowth without altering the number of cells in the choroid explant angiogenic assay, resulting in more densely packed outgrowth. Hcy prevented the outward orientation of actin filaments and decreased the number of actin projections along the leading edge of outgrowth. Hcy also slowed outgrowth from the edge of a scraped endothelial monolayer and in cultures of dispersed cells, Hcy impaired cell locomotion without affecting proliferation. Inhibition of iNOS activity rescued the effect of Hcy on area of explant outgrowth, cell density, number of projections, cell locomotion, and rate of outgrowth following scraping. CONCLUSIONS: Hcy impairs microvascular endothelial outgrowth, but not proliferation, by disrupting cell locomotion through an iNOS-dependent mechanism.

Opening of the blood-brain barrier before cerebral pathology in mild hyperhomocysteinemia.

Hyperhomocysteinemia (HHcy) is a risk factor for cognitive impairment. The purpose of this study was to determine the temporal pattern of cerebral pathology in a mouse model of mild HHcy, because understanding this time course provides the basis for understanding the mechanisms involved. C57Bl/6 mice with heterozygous deletion cystathionine ß-synthase (cbs (+/-); Het) were used as a model of mild HHcy along with their wild-type littermates (cbs (+/+); WT). Mice were 'young' (5.3±0.2 months of age) and 'old' (16.6±0.9 months of age). Blood-brain barrier (BBB) permeability was quantified from Evans blue and sodium fluorescein extravasation. Microvascular architecture was assessed by z-stack confocal microscopy. Leukoaraiosis was measured from Luxol fast blue stained slides of paraffin brain sections. Inflammation was quantified using standard antibody-based immunohistochemical techniques. Cognitive function was assessed using the Morris water maze. BBB permeability was significantly greater in Het vs. WT mice at all ages (p<0.05). There were no differences in microvascular architecture among the groups. Compared with all other groups, old Het mice had significantly greater leukoaraiosis, inflammation in the fornix, and cognitive impairment (p<0.05). In mild HHcy, increased permeability of the BBB precedes the onset of cerebral pathology. This new paradigm may play a role in the progression of disease in HHcy.

Hyperhomocysteinemic mice show cognitive impairment without features of Alzheimer's disease phenotype.

Hyperhomocysteinemia (HHcy) is associated with cognitive impairment and Alzheimer's disease. Whether this association is mechanistic remains unclear. Here, we used a mouse model to test the hypothesis that HHcy increases levels of amyloid-ß (Aß) transporters in microvessels that form the blood-brain barrier, elevates Aß content (Aß40 and Aß42) in the brain, and impairs cognitive performance. Mice with HHcy and age-matched, non-HHcy controls (Ctrl) were studied in two age groups: adult (6.2 ± 0.4 months of age) and old (19 ± 2.0 months of age). Levels of Aß transporters, RAGE, LRP1, and Pgp, were not different between HHcy and Ctrl mice. Though there was an increase in overall brain Aß levels with age, there were no differences between HHcy and Ctrl groups in cortex, hippocampus, or midbrain/diencephalon. Despite the lack of difference in Aß, old mice with HHcy showed significant cognitive impairment on Morris water maze tests compared with Ctrl mice. We conclude that HHcy leads to cognitive impairment without many of the changes currently thought to be relevant to promoting the AD phenotype.

Homocysteine impairs endothelial wound healing by activating metabotropic glutamate receptor 5.

OBJECTIVE: Hcy is an independent risk factor for cerebrovascular disease and cognitive impairment. The purpose of this study was to elucidate the role of mGluR5 in Hcy-mediated impairment of cerebral endothelial wound repair. METHODS: Mouse CMVECs (bEnd.3) were used in conjunction with directed pharmacology and shRNA. AutoDock was used to simulate the docking of ligand-receptor interactions. RESULTS: Hcy (20 µM) significantly increased Cx43-pS368 by mGluR5- and PKC-dependent mechanisms. Hcy attenuated wound repair by an mGluR5-dependent mechanism over the six-day study period but did not alter cell proliferation in a proliferation assay, suggesting that the attenuation of wound repair may be due to dysfunctional migration in HHcy. Hcy increased the expression of Cx43 and Cx43-pS368 at the wound edge by activating mGluR5. Direct activation of mGluR5, using the specific agonist CHPG, was sufficient to reproduce the results whereas KO of mGluR5 with shRNA, or inhibition with MPEP, blocked the response to Hcy. CONCLUSIONS: Inhibition of mGluR5 activation could be a novel strategy for promoting endothelial wound repair in patients with HHcy. Activation of mGluR5 may be a viable strategy for disrupting angiogenesis.

Metabotropic glutamate receptor 5 mediates phosphorylation of vascular endothelial cadherin and nuclear localization of ß-catenin in response to homocysteine.

Elevated plasma homocysteine (Hcy) is an independent risk factor for vascular disease and stroke in part by causing generalized endothelial dysfunction. A receptor that is sensitive to Hcy and its intracellular signaling systems has not been identified. ß-catenin is a pleiotropic regulator of transcription and cell function. Using a brain microvascular endothelial cell line (bEnd.3), we tested the hypothesis that Hcy causes receptor-dependent nuclear translocation of ß-catenin. Hcy increased phosphorylation of Y731 on vascular endothelial cadherin (VE-cadherin), a site involved in coupling ß-catenin to VE-cadherin. This was blocked by inhibition of either metabotropic glutamate receptor 5 (mGluR5) or ionotropic glutamate receptor (NMDAr) and by shRNA knockdown of mGluR5. Expression of these receptors was confirmed by flow cytometry, immunohistochemistry, and western blotting. Directed pharmacology with specific agonists elucidated a signaling cascade where Hcy activates mGluR5 which activates NMDAr with subsequent PKC activation and uncoupling of the VE-cadherin/ß-catenin complex. Moreover, Hcy caused a shift in localization of ß-catenin from membrane-bound VE-cadherin to the cell nucleus, where it bound DNA, including a regulatory region of the gene for claudin-5, leading to reduced expression of claudin-5. Nuclear localization, DNA binding of ß-catenin, and reduced claudin-5 expression were blocked by inhibition of mGluR5. Knockdown of mGluR5 expression with shRNA also rescued claudin-5 expression from the effects of Hcy treatment. These data uniquely identify mGluR5 as a master switch that drives ß-catenin nuclear localization in vascular endothelium and regulates cell-cell coupling in response to elevated Hcy levels. These studies dissect a pharmacological opportunity for developing new therapeutic strategies in HHcy.

Nitrative stress in cerebral endothelium is mediated by mGluR5 in hyperhomocysteinemia.

Hyperhomocysteinemia (HHcy) disrupts nitric oxide (NO) signaling and increases nitrative stress in cerebral microvascular endothelial cells (CMVECs). This is mediated, in part, by protein nitrotyrosinylation (3-nitrotyrosine; 3-NT) though the mechanisms by which extracellular homocysteine (Hcy) generates intracellular 3-NT are unknown. Using a murine model of mild HHcy (cbs(+/-) mouse), we show that 3-NT is significantly elevated in cerebral microvessels with concomitant reductions in serum NO bioavailability as compared with wild-type littermate controls (cbs(+/+)). Directed pharmacology identified a receptor-dependent mechanism for 3-NT formation in CMVECs. Homocysteine increased expression of inducible NO synthase (iNOS) and formation of 3-NT, both of which were blocked by inhibition of metabotropic glutamate receptor-5 (mGluR5) with the specific antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride. Activation of mGluR5 is both sufficient and necessary to drive the nitrative stress because direct activation using the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine also increased iNOS expression and 3-NT formation while knockdown of mGluR5 receptor expression by short hairpin RNA (shRNA) blocked their increase in response to Hcy. Nitric oxide derived from iNOS was required for Hcy-mediated formation of 3-NT because the effect was blocked by 1400W. These results provide the first evidence for a receptor-dependent process that explains how plasma Hcy levels control intracellular nitrative stress in cerebral microvascular endothelium.

Hyperhomocysteinemia increases permeability of the blood-brain barrier by NMDA receptor-dependent regulation of adherens and tight junctions.

Hyperhomocysteinemia (HHcy) increases permeability of the blood-brain barrier, but the mechanisms are undetermined. Homocysteine (Hcy) is an agonist of the neuronal N-methyl-D-aspartate receptor (NMDAr). We tested the hypothesis that HHcy disrupts the blood-brain barrier by an NMDAr-dependent mechanism in endothelium. In brain microvascular endothelial cells, there was no change in expression of the adherens junction protein VE-cadherin with Hcy treatment, but there was a significant decrease in the amount of ß-catenin at the membrane. Moreover, Hcy caused nuclear translocation of ß-catenin and attachment to the promoter for the tight junction protein claudin-5, with concomitant reduction in claudin-5 expression. Using a murine model of HHcy (cbs(+/-)), treatment for 2 weeks with an NMDAr antagonist (memantine) rescued cerebrovascular expression of claudin-5 and blood-brain barrier permeability to both exogenous sodium fluorescein and endogenous IgG. Memantine had no effect on these parameters in wild-type littermates. The same results were obtained using an in vitro model with brain microvascular endothelial cells. These data provide the first evidence that the NMDAr is required for Hcy-mediated increases in blood-brain barrier permeability. Modulating cerebral microvascular NMDAr activity may present a novel therapeutic target in diseases associated with opening of the blood-brain barrier in HHcy, such as stroke and dementia.

Vascular complications of cystathionine ß-synthase deficiency: future directions for homocysteine-to-hydrogen sulfide research.

Homocysteine (Hcy), a cardiovascular and neurovascular disease risk factor, is converted to hydrogen sulfide (H(2)S) through the transsulfuration pathway. H(2)S has attracted considerable attention in recent years for many positive effects on vascular health and homeostasis. Cystathionine ß-synthase (CBS) is the first, and rate-limiting, enzyme in the transsulfuration pathway. Mutations in the CBS gene decrease enzymatic activity, which increases the plasma Hcy concentration, a condition called hyperhomocysteinemia (HHcy). Animal models of CBS deficiency have provided invaluable insights into the pathological effects of transsulfuration impairment and of both mild and severe HHcy. However, studies have also highlighted the complexity of HHcy and the need to explore the specific details of Hcy metabolism in addition to Hcy levels per se. There has been a relative paucity of work addressing the dysfunctional H(2)S production in CBS deficiency that may contribute to, or even create, HHcy-associated pathologies. Experiments using CBS knockout mice, both homozygous (-/-) and heterozygous (+/-), have provided 15 years of new knowledge and are the focus of this review. These murine models present the opportunity to study a specific mechanism for HHcy that matches one of the etiologies in many human patients. Therefore, the goal of this review was to integrate and highlight the critical information gained thus far from models of CBS deficiency and draw attention to critical gaps in knowledge, with particular emphasis on the modulation of H(2)S metabolism. We include findings from human and animal studies to identify important opportunities for future investigation that should be aimed at generating new basic and clinical understanding of the role of CBS and transsulfuration in cardiovascular and neurovascular disease.

Extracellular transsulfuration generates hydrogen sulfide from homocysteine and protects endothelium from redox stress.

Homocysteine, a cardiovascular and neurocognitive disease risk factor, is converted to hydrogen sulfide, a cardiovascular and neuronal protectant, through the transsulfuration pathway. Given the damaging effects of free homocysteine in the blood and the importance of blood homocysteine concentration as a prognosticator of disease, we tested the hypotheses that the blood itself regulates homocysteine-hydrogen sulfide metabolism through transsulfuration and that transsulfuration capacity and hydrogen sulfide availability protect the endothelium from redox stress. Here we show that the transsulfuration enzymes, cystathionine ß-synthase and cystathionine γ-lyase, are secreted by microvascular endothelial cells and hepatocytes, circulate as members of the plasma proteome, and actively produce hydrogen sulfide from homocysteine in human blood. We further demonstrate that extracellular transsulfuration regulates cell function when the endothelium is challenged with homocysteine and that hydrogen sulfide protects the endothelium from serum starvation and from hypoxia-reoxygenation injury. These novel findings uncover a unique set of opportunities to explore innovative clinical diagnostics and therapeutic strategies in the approach to homocysteine-related conditions such as atherosclerosis, thrombosis, and dementia.

Regional activation of rapid onset vasodilatation in mouse skeletal muscle: regulation through α-adrenoreceptors.

Exercise onset entails motor unit recruitment and the initiation of vasodilatation. Dilatation can ascend the arteriolar network to encompass proximal feed arteries but is opposed by sympathetic nerve activity, which promotes vasoconstriction and inhibits ascending vasodilatation through activating α-adrenoreceptors. Whereas contractile activity can antagonize sympathetic vasoconstriction, more subtle aspects of this interaction remain to be defined. We tested the hypothesis that constitutive activation of α-adrenoreceptors governs blood flow distribution within individual muscles. The mouse gluteus maximus muscle (GM) consists of Inferior and Superior regions. Each muscle region is supplied by its own motor nerve and feed artery with an anastomotic arteriole (resting diameter 25 microm) that spans both muscle regions. In anaesthetized male C57BL/6J mice (3-5 months old), the GM was exposed and superfused with physiological saline solution (35 degrees C; pH 7.4). Stimulating the inferior gluteal motor nerve (0.1 ms pulse, 100 Hz for 500 ms) evoked a brief tetanic contraction and produced rapid (<1 s) onset vasodilatation (ROV; diameter change, 10 +/- 1 µm) of the anastomotic arteriole along the active (Inferior) muscle region but not along the inactive (Superior) region (n = 8). In contrast, microiontophoresis of acetylcholine (1 µm micropipette tip, 1 µA, 500 ms) initiated dilatation that travelled along the anastomotic arteriole from the Inferior into the Superior muscle region (diameter change, 5 +/- 2 µm). Topical phentolamine (1 µm) had no effect on resting diameter but this inhibition of α-adrenoreceptors enabled ROV to spread along the anastomotic arteriole into the inactive muscle region (dilatation, 7 +/- 1 µm; P < 0.05), where remote dilatation to acetylcholine then doubled (P < 0.05). These findings indicate that constitutive activation of α-adrenoreceptors in skeletal muscle can restrict the spread of dilatation within microvascular resistance networks and thereby increase blood flow to active muscle regions.

Hypoxic pulmonary vasodilation: a paradigm shift with a hydrogen sulfide mechanism.

Hypoxic pulmonary vasoconstriction (HVC), an intrinsic and assumed ubiquitous response of mammalian pulmonary blood vessels, matches regional ventilation to perfusion via an unknown O(2)-sensing mechanism. Global pulmonary hypoxia experienced by individuals suffering from chronic obstructive pulmonary disease or numerous hypoventilation syndromes, including sleep apnea, often produces maladaptive pulmonary hypertension, but pulmonary hypertension is not observed in diving mammals, where profound hypoxia is routine. Here we examined the response of cow and sea lion pulmonary arteries (PA) to hypoxia and observed the expected HVC in the former and a unique hypoxic vasodilation in resistance vessels in the latter. We then used this disparate response to examine the O(2)-sensing mechanism. In both animals, exogenous H(2)S mimicked the vasoactive effects of hypoxia in isolated PA. H(2)S-synthesizing enzymes, cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfur transferase, were identified in lung tissue from both animals by one-dimensional Western blot analysis and immunohistochemistry. The relationship between H(2)S production/consumption and O(2) was examined in real time by use of amperometric H(2)S and O(2) sensors. H(2)S was produced by sea lion and cow lung homogenate in the absence of O(2), but it was rapidly consumed when O(2) was present. Furthermore, consumption of exogenous H(2)S by cow lung homogenate, PA smooth muscle cells, and heart mitochondria was O(2) dependent and exhibited maximal sensitivity at physiologically relevant Po(2) levels. These studies show that HVC is not an intrinsic property of PA and provide further evidence for O(2)-dependent H(2)S metabolism in O(2) sensing.