Chronic stress physically spares but functionally impairs innate-like invariant T cells.

The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a "split" inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.

Stress-elicited glucocorticoid receptor signaling upregulates TIGIT in innate-like invariant T lymphocytes.

Stress is known to impede certain host defense mechanisms, including those governed by conventional T lymphocytes. However, whether innate-like T lymphocytes, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, are impacted by stress is unclear. Herein, we report that prolonged psychological stress caused by physical confinement results in robust upregulation of T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), an immune checkpoint receptor that controls antitumor and antiviral immune responses. Elevated TIGIT expression was found not only on NK and conventional T cells, but also on iNKT and MAIT cells. Stress-provoked TIGIT upregulation was reversed through treatment with the glucocorticoid receptor (GR) antagonist RU486, but not with 6-hydroxydopamine that induces chemical sympathectomy. A Cre/Lox gene targeting model in which GR was ablated in cells expressing Lck under its proximal promoter revealed that TIGIT upregulation in stressed animals stems from direct GR signaling in T and iNKT cells. In fact, long-term oral administration of exogenous corticosterone (CS) to wild-type C57BL/6 (B6) mice was sufficient to increase TIGIT expression levels on T and iNKT cells. In vitro treatment with CS also potently and selectively upregulated TIGIT, but not CTLA-4 or LAG-3, on mouse iNKT and MAIT hybridomas. These results were recapitulated using primary hepatic iNKT and MAIT cells from wild-type B6 and B6.MAITCAST mice, respectively. Subjecting B6.MAITCAST mice to physical restraint also raised the frequency of TIGIT+ cells among hepatic MAIT cells in a GR-dependent manner. Finally, we found that TIGIT is similarly upregulated in a chronic variable stress model in which animals are exposed to unpredictable heterotypic stressors without developing habituation. Taken together, our findings link, for the first time to our knowledge, GR signaling to TIGIT expression. We propose that glucocorticoid hormones dampen immune responses, in part, by enhancing TIGIT expression across multiple critical subsets of effector lymphocytes, including innate-like T cells. Therefore, TIGIT may constitute an attractive target in immune-enhancing interventions for sustained physiological stress.

A streak length-based method for quantifying red blood cell flow in skeletal muscle arteriolar networks.

OBJECTIVE: To develop an experimental method to quantify RBC flow throughout skeletal muscle arteriolar networks. METHODS: Data on arteriolar geometry were obtained using IVVM of the rat GM. RBC velocities and number densities were also obtained during these experiments using fluorescently labeled RBCs. Arteriolar and RBC data were combined to estimate blood volume flow rates, HT and HD values, and RBC volume flow rates. Validation of hematocrit and RBC flow results was performed at arteriolar bifurcations using both mass balance and comparisons to an established model of the PS effect. RESULTS: Estimated HT values were within the expected range (6%-34%) for the arterioles considered (29-130 µm). RBC mass balance error was 18 ± 16% (mean ± SD, n = 7 bifurcations). RBC outflow from diverging bifurcations as a function of RBC inflow was given by Y = 0.986*X + 0.331 with R2  = 0.987. Outflow HT as a function of the PS prediction was given by Y = 1.034*X + 0.004 with R2  = 0.691. RBC outflow as a function of the prediction was given by Y = 0.917*X + 0.804 with R2  = 0.891. CONCLUSIONS: An experimental method has been developed and validated that can easily and accurately quantify RBC flow distribution in large skeletal muscle arteriolar networks and provides direct estimates of HT values.

Neuropeptide Y1 and alpha-1 adrenergic receptor-mediated decreases in functional vasodilation in gluteus maximus microvascular networks of prediabetic mice.

Prediabetes is associated with impaired contraction-evoked dilation of skeletal muscle arterioles, which may be due to increased sympathetic activity accompanying this early stage of diabetes disease. Herein, we sought to determine whether blunted contraction-evoked vasodilation resulted from enhanced sympathetic neuropeptide Y1 receptor (Y1R) and alpha-1 adrenergic receptor (α1R) activation. Using intravital video microscopy, second-, third-, and fourth-order (2A, 3A, and 4A) arteriolar diameters were measured before and following electrical field stimulation of the gluteus maximus muscle (GM) in prediabetic (PD, Pound Mouse) and control (CTRL, c57bl6, CTRL) mice. Baseline diameter was similar between groups; however, single tetanic contraction (100 Hz; 400 and 800 msec) and sustained rhythmic contraction (2 and 8 Hz, 30 sec) evoked rapid onset vasodilation and steady-state vasodilatory responses that were blunted by 50% or greater in PD versus CTRL. Following Y1R and α1R blockade with sympathetic antagonists BIBP3226 and prazosin, contraction-evoked arteriolar dilation in PD was restored to levels observed in CTRL. Furthermore, arteriolar vasoconstrictor responses to NPY (10-13 -10-8  mol/L) and PE (10-9 -10-5  mol/L) were greater in PD versus CTRL at higher concentrations, especially at 3A and 4A. These findings suggest that contraction-evoked vasodilation in PD is blunted by Y1R and α1R receptor activation throughout skeletal muscle arteriolar networks.

Beneficial Pleiotropic Antidepressive Effects of Cardiovascular Disease Risk Factor Interventions in the Metabolic Syndrome.

BACKGROUND: Although the increased prevalence and severity of clinical depression and elevated cardiovascular disease risk represent 2 vexing public health issues, the growing awareness of their combined presentation compounds the challenge. The obese Zucker rat, a model of the metabolic syndrome, spontaneously develops significant depressive symptoms in parallel with the progression of the metabolic syndrome and, thus, represents a compelling model for study. The primary objective was to assess the impact on both cardiovascular outcomes, specifically vascular structure and function, and depressive symptoms in obese Zucker rats after aggressive treatment for cardiovascular disease risk factors with long-term exercise or targeted pharmacological interventions. METHODS AND RESULTS: We chronically treated obese Zucker rats with clinically relevant interventions against cardiovascular disease risk factors to determine impacts on vascular outcomes and depressive symptom severity. While most of the interventions (chronic exercise, anti-hypertensive, the interventions (long-term exercise, antihypertensive, antidyslipidemia, and antidiabetic) were differentially effective at improving vascular outcomes, only those that also resulted in a significant improvement to oxidant stress, inflammation, arachidonic acid metabolism (prostacyclin versus thromboxane A2), and their associated sequelae were effective at also blunting depressive symptom severity. Using multivariable analyses, discrimination between the effectiveness of treatment groups to maintain behavioral outcomes appeared to be dependent on breaking the cycle of inflammation and oxidant stress, with the associated outcomes of improving endothelial metabolism and both cerebral and peripheral vascular structure and function. CONCLUSIONS: This initial study provides a compelling framework from which to further interrogate the links between cardiovascular disease risk factors and depressive symptoms and suggests mechanistic links and potentially effective avenues for intervention.

Protection from chronic stress- and depressive symptom-induced vascular endothelial dysfunction in female rats is abolished by preexisting metabolic disease.

While it is known that chronic stress and clinical depression are powerful predictors of poor cardiovascular outcomes, recent clinical evidence has identified correlations between the development of metabolic disease and depressive symptoms, creating a combined condition of severely elevated cardiovascular disease risk. In this study, we used the obese Zucker rat (OZRs) and the unpredictable chronic mild stress (UCMS) model to determine the impact of preexisting metabolic disease on the relationship between chronic stress/depressive symptoms and vascular function. Additionally, we determined the impact of metabolic syndrome on sex-based protection from chronic stress/depressive effects on vascular function in female lean Zucker rats (LZRs). In general, vasodilator reactivity was attenuated under control conditions in OZRs compared with LZRs. Although still impaired, conduit arterial and resistance arteriolar dilator reactivity under control conditions in female OZRs was superior to that in male or ovariectomized (OVX) female OZRs, largely because of better maintenance of vascular nitric oxide and prostacyclin levels. However, imposition of metabolic syndrome in combination with UCMS in OZRs further impaired dilator reactivity in both vessel subtypes to a similarly severe extent and abolished any protective effect in female rats compared with male or OVX female rats. The loss of vascular protection in female OZRs with UCMS was reflected in vasodilator metabolite levels, which closely matched those in male and OVX female OZRs subjected to UCMS. These results suggest that presentation of metabolic disease in combination with depressive symptoms can overwhelm the vasoprotection identified in female rats and, thereby, may reflect a severe impairment to normal endothelial function. NEW & NOTEWORTHY This study addresses the protection from chronic stress- and depression-induced vascular dysfunction identified in female compared with male or ovariectomized female rats. We determined the impact of preexisting metabolic disease, a frequent comorbidity of clinical depression in humans, on that vascular protection. With preexisting metabolic syndrome, female rats lost all protection from chronic stress/depressive symptoms and became phenotypically similar to male and ovariectomized female rats, with comparably poor vasoactive dilator metabolite profiles.

Protection from vascular dysfunction in female rats with chronic stress and depressive symptoms.

The increasing prevalence and severity of clinical depression are strongly correlated with vascular disease risk, creating a comorbid condition with poor outcomes but demonstrating a sexual disparity whereby female subjects are at lower risk than male subjects for subsequent cardiovascular events. To determine the potential mechanisms responsible for this protection against stress/depression-induced vasculopathy in female subjects, we exposed male, intact female, and ovariectomized (OVX) female lean Zucker rats to the unpredictable chronic mild stress (UCMS) model for 8 wk and determined depressive symptom severity, vascular reactivity in ex vivo aortic rings and middle cerebral arteries (MCA), and the profile of major metabolites regulating vascular tone. While all groups exhibited severe depressive behaviors from UCMS, severity was significantly greater in female rats than male or OVX female rats. In all groups, endothelium-dependent dilation was depressed in aortic rings and MCAs, although myogenic activation and vascular (MCA) stiffness were not impacted. Higher-resolution results from pharmacological and biochemical assays suggested that vasoactive metabolite profiles were better maintained in female rats with normal gonadal sex steroids than male or OVX female rats, despite increased depressive symptom severity (i.e., higher nitric oxide and prostacyclin and lower H2O2 and thromboxane A2 levels). These results suggest that female rats exhibit more severe depressive behaviors with UCMS but are partially protected from the vasculopathy that afflicts male rats and female rats lacking normal sex hormone profiles. Determining how female sex hormones afford partial vascular protection from chronic stress and depression is a necessary step for addressing the burden of these conditions on cardiovascular health. NEW & NOTEWORTHY This study used a translationally relevant model for chronic stress and elevated depressive symptoms to determine how these factors impact conduit and resistance arteriolar function in otherwise healthy rats. While chronic stress leads to an impaired vascular reactivity associated with elevated oxidant stress, inflammation, and reduced metabolite levels, we demonstrated partial protection from vascular dysfunction in female rats with normal sex hormone profiles compared with male or ovariectomized female rats.

Endogenous dipeptidyl peptidase IV modulates skeletal muscle arteriolar diameter in rats.

The purpose of this study is to investigate that dipeptidyl peptidase IV (DPP-IV) released from skeletal and vascular smooth muscle can increase arteriolar diameter in a skeletal muscle vascular bed by reducing neuropeptide Y (NPY)-mediated vasoconstriction. We hypothesized that the effect of myokine DPP-IV would be greatest in the smallest and least in the largest arterioles. Eight male Sprague Dawley rats (age 7-9 weeks; mass, mean ± SD: 258 ± 41 g) were anesthetized and the gluteus maximus dissected in situ for intravital microscopy analysis of arteriolar diameter of the vascular network. Computational modeling was performed on the diameter measurements to evaluate the overall impact of diameter changes on network resistance and flow distribution. In the first set of experiments, whey protein isolate powder was added to physiological saline solution, put in a heated reservoir, and applied to the preparation to induce release of DPP-IV from the muscle. This resulted in an order-dependent increase in arteriolar diameter, with the largest change in the 6A arterioles (63% more reactive than 1A arterioles; P < 0.05). This effect was abolished by adding the DPP-IV inhibitor, Diprotin A. To test if the DPP-IV released was affecting NPY-mediated vasoconstriction, we applied NPY and whey protein, which resulted in attenuated vasoconstriction. These findings suggest that DPP-IV is released from muscle and has a unique effect on blood flow, which appears to act on NPY to attenuate vasoconstriction. The findings suggest that DPP-IV released from the skeletal or smooth muscle can alter muscle blood flow.

Estimating blood flow in skeletal muscle arteriolar trees reconstructed from in vivo data using the Fry approach.

OBJECTIVE: To develop a computational method to accurately predict blood flow in skeletal muscle arteriolar trees in the absence of complete boundary data. METHODS: We used arteriolar trees in the rat GM muscle that were reconstructed from montages obtained via IVVM, and incorporated a recently published method for approximating unknown b.c.'s into our existing two-phase, steady-state blood flow model. For varying numbers of unknown b.c.'s, we used the new flow model and GM geometry to approximately match RBC flows corresponding to experimental measurements. RESULTS: We showed this method gives errors that decrease as the number of unknown b.c.'s decreases. We also showed that specifying total blood flow decreases the mean RBC flow error and its variability. By varying required target values of intravascular pressure and wall shear stress, we showed results are less sensitive to target pressure. Finally, we developed and validated a method for determining target values, so that network hemodynamics and resistance can be accurately calculated based only on measured or estimated total blood flow. CONCLUSIONS: We have developed and validated a computational method that can accurately estimate RBC flow distribution in skeletal muscle arteriolar trees in the absence of complete boundary data.

Altered post-capillary and collecting venular reactivity in skeletal muscle with metabolic syndrome.

KEY POINTS: With the development of the metabolic syndrome, both post-capillary and collecting venular dilator reactivity within the skeletal muscle of obese Zucker rats (OZR) is impaired. The impaired dilator reactivity in OZR reflects a loss in venular nitric oxide and PGI2 bioavailability, associated with the chronic elevation in oxidant stress. Additionally, with the impaired dilator responses, a modest increase in adrenergic constriction combined with an elevated thromboxane A2 production may contribute to impaired functional dilator and hyperaemic responses at the venular level. For the shift in skeletal muscle venular function with development of the metabolic syndrome, issues such as aggregate microvascular perfusion resistance, mass transport and exchange within with capillary networks, and fluid handling across the microcirculation are compelling avenues for future investigation. ABSTRACT: While research into vascular outcomes of the metabolic syndrome has focused on arterial/arteriolar and capillary levels, investigation into venular function and how this impacts responses has received little attention. Using the in situ cremaster muscle of obese Zucker rats (OZR; with lean Zucker rats (LZR) as controls), we determined indices of venular function. At ∼17 weeks of age, skeletal muscle post-capillary venular density was reduced by ∼20% in LZR vs. OZR, although there was no evidence of remodelling of the venular wall. Venular tone at ∼25 µm (post-capillary) and ∼75 µm (collecting) diameter was elevated in OZR vs. LZR. Venular dilatation to acetylcholine was blunted in OZR vs. LZR due to increased oxidant stress-based loss of nitric oxide bioavailability (post-capillary) and increased α1 - (and α2 -) mediated constrictor tone (collecting). Venular constrictor responses in OZR were comparable to LZR for most stimuli, although constriction to α1 -adrenoreceptor stimulation was elevated. In response to field stimulation of the cremaster muscle (0.5, 1, 3 Hz), venular dilator and hyperaemic responses to lower frequencies were blunted in OZR, but responses at 3 Hz were similar between strains. Venous production of TxA2 was higher in OZR than LZR and significantly higher than PGI2 production in either following arachidonic acid challenge. These results suggest that multi-faceted alterations to skeletal muscle venular function in OZR may contribute to alterations in upstream capillary pressure profiles and the transcapillary exchange of solutes and water under conditions of metabolic syndrome.

Comprehensive In Situ Analysis of Arteriolar Network Geometry and Topology in Rat Gluteus Maximus Muscle.

OBJECTIVES: To provide detailed geometric and topological descriptions of the rat gluteus maximus arteriolar network, and to measure the distribution of diameters and lengths as well as their associated variability within and between networks. METHODS: Complete arteriolar networks arising from feed artery (inferior gluteal artery) to terminal branches were imaged under baseline conditions, using IVVM. Photomontages of complete networks were assembled and evaluated offline for measurements of geometry and topology. Single-line (skeletonized) tracings of the networks were made for fractal analysis. RESULTS: Diameters and lengths decreased with increasing topological order (centrifugal), while number of elements increased with increasing order. Horton's laws were shown to be valid within the arteriolar networks of the rat GM. Inter-network variability in diameter (~5-22%) and length (~17-30%) at each order was generally lower than the corresponding intra-network variability in diameter (~10-48%) and length (~39-106%). CONCLUSIONS: Data presented in this study provide crucial quantitative analysis of complete arteriolar networks within healthy skeletal muscle, and may serve as ideal experimental inputs for future theoretical studies of skeletal muscle microvascular structure and function.

A Microvascular Wall Shear Rate Function Derived From In Vivo Hemodynamic and Geometric Parameters in Continuously Branching Arterioles.

OBJECTIVES: Conventional approaches to WSR estimation in the microcirculation involve assumptions that may result in under-/over-estimation of WSR. Therefore, our objectives were: (i) calculate WSR from RBC velocity profiles for a wide range of arteriolar diameters, (ii) provide an experimentally derived and straightforward WSR estimation function, and (iii) compare calculated to conventional WSR estimations. METHODS: We characterized RBC velocity profiles in arterioles (n = 39) of branching networks (21-115 µm) in the rat gluteus maximus muscle (n = 6). Measures included mean and maximum velocities, CFL thickness, and RBC column edge velocity, and an experiment-based WSR function was derived. RESULTS: CFL thickness (1-4.3 µm) positively correlated with arteriolar diameter (r(2)  = 0.64). Results from the WSR equation were similar to values from edge RBC velocities/CFL. Experimental WSRs (1317-4334/sec) were independent of arteriolar diameter, and were greater than pseudoshear rates (for VRatio of 1.6, 2, or diameter-dependent VRatio function) (p < 0.05). CONCLUSION: A WSR equation was derived from experimental hemodynamic parameters, and is adaptable to other velocity measurement techniques in order to obtain WSR and stress (when plasma viscosity is known). These findings provide insight on the nature of conventional WSR calculation methods in underestimating microvascular WSR values.

From one generation to the next: a comprehensive account of sympathetic receptor control in branching arteriolar trees.

The effect of the sympathetic nervous system on blood flow distribution within skeletal muscle microvasculature is conditional upon regional activation of receptors for sympathetic neurotransmitters. Previous studies have shown that proximal arterioles are largely governed by adrenergic activation, whereas it is speculated that distal branches are controlled by peptidergic and purinergic activation. However, no study has systematically evaluated the activation of adrenergic, peptidergic and purinergic receptors in continuously branching arteriolar trees of an individual skeletal muscle model. Therefore, in the present study, sympathetic agonists were used to evaluate the constriction responses along first to fifth order arterioles in continuously branching arteriolar trees of a in vivo rat gluteus maximus muscle preparation with respect to specific activation of receptors for sympathetic neurotransmitters (α1R, α2R, NPY1R and P2X1R). Constriction responses were incorporated into a mathematical blood flow model to estimate the total flow, resistance and red blood cell flow heterogeneity within a computationally reconstructed gluteus maximus arteriolar network. For the first time, the effects of activating receptors for sympathetic neurotransmitters on vasoconstrictor responses and the ensuing haemodynamics in continuously branching arteriolar trees of skeletal muscle were characterized, where proximal arterioles responded most to α1R and α2R adrenergic activation, whereas distal arterioles responded most to Y1R and P2X1R activation. Total flow and resistance changed with activation of all receptors, whereas red blood cell flow heterogeneity was largely affected by peptidergic and purinergic activation in distal arterioles. The reported data highlight the functional consequences of topologically-dependent sympathetic control and may serve as novel input parameters in computational modelling of network flow.

Exercise training enhances insulin-stimulated nerve arterial vasodilation in rats with insulin-treated experimental diabetes.

Insulin stimulates nerve arterial vasodilation through a nitric oxide (NO) synthase (NOS) mechanism. Experimental diabetes reduces vasa nervorum NO reactivity. Studies investigating hyperglycemia and nerve arterial vasodilation typically omit insulin treatment and use sedentary rats resulting in severe hyperglycemia. We tested the hypotheses that 1) insulin-treated experimental diabetes and inactivity (DS rats) will attenuate insulin-mediated nerve arterial vasodilation, and 2) deficits in vasodilation in DS rats will be overcome by concurrent exercise training (DX rats; 75-85% VO2 max, 1 h/day, 5 days/wk, for 10 wk). The baseline index of vascular conductance values (VCi = nerve blood flow velocity/mean arterial blood pressure) were similar (P ≥ 0.68), but peak VCi and the area under the curve (AUCi) for the VCi during a euglycemic hyperinsulinemic clamp (EHC; 10 mU·kg(-1)·min(-1)) were lower in DS rats versus control sedentary (CS) rats and DX rats (P ≤ 0.01). Motor nerve conduction velocity (MNCV) was lower in DS rats versus CS rats and DX rats (P ≤ 0.01). When compared with DS rats, DX rats expressed greater nerve endothelial NOS (eNOS) protein content (P = 0.04). In a separate analysis, we examined the impact of diabetes in exercise-trained rats alone. When compared with exercise-trained control rats (CX), DX rats had a lower AUCi during the EHC, lower MNCV values, and lower sciatic nerve eNOS protein content (P ≤ 0.03). Therefore, vasa nervorum and motor nerve function are impaired in DS rats. Such deficits in rats with diabetes can be overcome by concurrent exercise training. However, in exercise-trained rats (CX and DX groups), moderate hyperglycemia lowers vasa nervorum and nerve function.

Neuropeptide Y Y5-receptor activation on breast cancer cells acts as a paracrine system that stimulates VEGF expression and secretion to promote angiogenesis.

Accumulating data implicate a pathological role for sympathetic neurotransmitters like neuropeptide Y (NPY) in breast cancer progression. Our group and others reported that NPY promotes proliferation and migration in breast cancer cells, however the angiogenic potential of NPY in breast cancer is unknown. Herein we sought to determine if NPY promotes angiogenesis in vitro by increasing vascular endothelial growth factor (VEGF) expression and release from 4T1 breast cancer cells. Western blot analysis revealed that NPY treatment caused a 52 ± 14% increase in VEGF expression in the 4T1 cells compared to non-treated controls. Using selective NPY Y-receptor agonists (Y1R, Y2R and Y5R) we observed an increase in VEGF expression only when cells were treated with Y5R agonist. Congruently, using selective Y1R, Y2R, or Y5R antagonists, NPY-induced increases in VEGF expression in 4T1 cells were attenuated only under Y5R antagonism. Endothelial tube formation assays were conducted using conditioned media (CM) from NPY treated 4T1 cells. Concentration-dependent increases in number of branch points and complete endothelial networks were observed in HUVEC exposed to NPY CM. CM from Y5R agonist treated 4T1 cells caused similar increases in number of branch points and complete endothelial networks. VEGF concentration was quantified in CM (ELISA) from agonist experiments; we observed a 2-fold and 2.5-fold increase in VEGF release from NPY and Y5R agonist treated 4T1 cells respectively. Overall these data highlight a novel mechanism by which NPY may promote breast cancer progression, and further implicate a pathological role of the NPY Y5R.

Effect of extraluminal ATP application on vascular tone and blood flow in skeletal muscle: implications for exercise hyperemia.

During skeletal muscle contractions, the concentration of ATP increases in muscle interstitial fluid as measured by microdialysis probes. This increase is associated with the magnitude of blood flow, suggesting that interstitial ATP may be important for contraction-induced vasodilation. However, interstitial ATP has solely been described to induce vasoconstriction in skeletal muscle. To examine whether interstitial ATP induces vasodilation in skeletal muscle and to what extent this vasoactive effect is mediated by formation of nitric oxide (NO) and prostanoids, three different experimental models were studied. The rat gluteus maximus skeletal muscle model was used to study changes in local skeletal muscle hemodynamics. Superfused ATP at concentrations found during muscle contractions (1-10 µM) increased blood flow by up to 400%. In this model, the underlying mechanism was also examined by inhibition of NO and prostanoid formation. Inhibition of these systems abolished the vasodilator effect of ATP. Cell-culture experiments verified ATP-induced formation of NO and prostacyclin in rat skeletal muscle microvascular endothelial cells, and ATP-induced formation of NO in rat skeletal muscle cells. To confirm these findings in humans, ATP was infused into skeletal muscle interstitium of healthy subjects via microdialysis probes and found to increase muscle interstitial concentrations of NO and prostacyclin by ~60% and ~40%, respectively. Collectively, these data suggest that a physiologically relevant elevation in interstitial ATP concentrations increases muscle blood flow, indicating that the contraction-induced increase in skeletal muscle interstitial [ATP] is important for exercise hyperemia. The vasodilator effect of ATP application is mediated by NO and prostanoid formation.

Pre-diabetes augments neuropeptide Y1- and α1-receptor control of basal hindlimb vascular tone in young ZDF rats.

BACKGROUND: Peripheral vascular disease in pre-diabetes may involve altered sympathetically-mediated vascular control. Thus, we investigated if pre-diabetes modifies baseline sympathetic Y(1)-receptor (Y(1)R) and α(1)-receptor (α(1)R) control of hindlimb blood flow (Q(fem)) and vascular conductance (VC). METHODS: Q(fem) and VC were measured in pre-diabetic ZDF rats (PD) and lean controls (CTRL) under infusion of BIBP3226 (Y(1)R antagonist), prazosin (α(1)R antagonist) and BIBP3226+prazosin. Neuropeptide Y (NPY) concentration and Y(1)R and α(1)R expression were determined from hindlimb skeletal muscle samples. RESULTS: Baseline Q(fem) and VC were similar between groups. Independent infusions of BIBP3226 and prazosin led to increases in Q(fem) and VC in CTRL and PD, where responses were greater in PD (p<0.05). The percent change in VC following both drugs was also greater in PD compared to CTRL (p<0.05). As well, Q(fem) and VC responses to combined blockade (BIBP3226+prazosin) were greater in PD compared to CTRL (p<0.05). Interestingly, an absence of synergistic effects was observed within groups, as the sum of the VC responses to independent drug infusions was similar to responses following combined blockade. Finally, white and red vastus skeletal muscle NPY concentration, Y(1)R expression and α(1)R expression were greater in PD compared to CTRL. CONCLUSIONS: For the first time, we report heightened baseline Y(1)R and α(1)R sympathetic control of Q(fem) and VC in pre-diabetic ZDF rats. In support, our data suggest that augmented sympathetic ligand and receptor expression in pre-diabetes may contribute to vascular dysregulation.

Y1R control of sciatic nerve blood flow in the Wistar Kyoto rat.

We hypothesized that neuropeptide Y (NPY) exerts vasoconstrictor properties in sciatic nerve blood supply by a Y1 receptor (Y1R) mechanism. Using Doppler ultrasound (40MHz), we measured blood flow velocity through a sciatic nerve supply artery during infusions of NPY and/or Y1R blockade with BIBP3226 in Wistar Kyoto rats before, and following, ganglionic blockade with Hexamethonium (Hex). Following Hex infusion, mean arterial pressure (baseline: 83±18, Hex: 57±3 mm Hg) was reduced. After 30 min, the index of conductance at the sciatic nerve (velocity/MAP expressed as % baseline) started to increase from 103±35 to 127±39% baseline in the following 30 min (p<0.05). Infusion of NPY (Y1 agonist) minimized this dilatory response (Hex baseline: 99±15, NPY: 104±11% baseline; NS). This NPY-induced attenuation was, in turn, minimized by BIBP3226 (Hex baseline: 73±12, NPY+BIBP3226: 89±14% baseline). Neither NPY nor BIBP3226 infusions without Hex affected the sciatic nerve arterial conductance. We conclude that the late dilation following Hex which is reversed by Y1R activation suggests some level of sympathetic control over sciatic nerve blood flow.

A simple "streak length method" for quantifying and characterizing red blood cell velocity profiles and blood flow in rat skeletal muscle arterioles.

OBJECTIVES: To develop a valid experimental method for quantifying blood flow in continuously branching skeletal muscle arterioles, and to derive an empirical relationship between velocity ratio (V(Max)/V(Mean)) and arteriolar diameter. METHODS: We evaluated arteriolar trees using IVVM of rat gluteus maximus muscle and developed a method to acquire single fluorescent-labeled RBC velocities across arteriolar lumens to create velocity profiles. These data were used to calculate the blood flow for 37 vessel segments (diameters: 21-115 µm). RESULTS: Mass balance at arteriolar bifurcations had 0.6 ± 3.2% error. Velocity ratios ranged from 1.35 to 1.98 and were positively correlated with diameter (p < 0.0001), and V(RBC) profiles were blunted with decreasing diameter. CONCLUSIONS: We present a means for quantifying blood flow in continuously branching skeletal muscle arterioles. Further, we provide an equation for calculating velocity ratios based on arteriolar diameter, which may be used by others for blood flow calculations.

Neuropeptide Y stimulates proliferation and migration in the 4T1 breast cancer cell line.

Stress has long been thought of to be associated with increased risk of cancer. Chronic stress is associated with elevated levels of sympathetic neurotransmitter (norepinephrine and neuropeptide Y: NPY) release and immunosuppression. The expression of NPY receptors has been reported in human breast carcinomas. Recently, activation of the NPY Y5 receptor was shown to stimulate cell growth and increase migration in human breast cancer cells; however the effects of NPY have yet to be investigated in a murine model of breast cancer. Thus, the specific aims of the current study were to: (i) characterize NPY receptor expression in 4T1 breast cancer cells and orthotopic tumors grown in BALB/c mice and (ii) investigate the impact of NPY receptor activation on 4T1 cell proliferation and migration in vitro. Positive expression of NPY receptors (Y1R, Y2R and Y5R) was observed in cells and tumor tissue. As well, NPY treatment of 4T1 cells promoted a concentration-dependent increase in proliferation, through increased phosphorylation of ERK 1/2. Using NPY receptor antagonists (Y1R:BIBP3226, Y2R:BIIE0246 and Y5R:L-152,804), we found the proliferative response to be Y5R mediated. Additionally, NPY increased chemotaxis through Y2R and Y5R activation. These data are in congruence with those from human cell lines and highlight the 4T1 cell line as a translatable model of breast cancer in which the effects of NPY can be studied in an immunocompetent system.