Dwayne N. Jackson

Neuropeptide Y and neurovascular control in skeletal muscle and skin

Neuropeptide Y (NPY) is a ubiquitous peptide with multiple effects on energy metabolism, reproduction, neurogenesis, and emotion. In addition, NPY is an important sympathetic neurotransmitter involved in neurovascular regulation. Although early studies suggested that the vasoactive effects of NPY were limited to periods of high stress, there is growing evidence for the involvement of NPY on baseline vasomotor tone and sympathetically evoked vasoconstriction in vivo in both skeletal muscle and the cutaneous circulation. In Sprague-Dawley rat skeletal muscle, Y(1)-receptor activation appears to play an important role in the regulation of basal vascular conductance, and this effect is similar in magnitude to the alpha(1)-receptor contribution. Furthermore, under baseline conditions, agonist and receptor-based mechanisms for Y(1)-receptor-dependent control of vascular conductance in skeletal muscle are greater in male than female rats. In skin, there is Y(1)-receptor-mediated vasoconstriction during whole body, but not local, cooling. As with the NPY system in muscle, this neural effect in skin differs between males and females and in addition, declines with aging. Intriguingly, skin vasodilation to local heating also requires NPY and is currently thought to be acting via a nitric oxide pathway. These studies are establishing further interest in the role of NPY as an important vasoactive agent in muscle and skin, adding to the complexity of neurovascular regulation in these tissues. In this review, we focus on the role of NPY on baseline vasomotor tone in skeletal muscle and skin and how NPY modulates vasomotor tone in response to stress, with the aim of compiling what is currently known, while highlighting some of the more pertinent questions yet to be answered.

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.

Gender‐modulated endogenous baseline neuropeptide Y Y1‐receptor activation in the hindlimb of Sprague‐Dawley rats

This study examined the effect of neuropeptide Y Y1‐receptor blockade both alone, and in interaction with α1‐adrenoceptor antagonism, on basal hindlimb vascular conductance in male and female Sprague‐Dawley rats. Hindlimb vascular conductance was measured during infusion of BIBP3226 (Y1‐receptor antagonist; 100 μg kg−1), prazosin (α1‐receptor antagonist; 20 μg kg−1), and combined blockade. In males, vascular conductance increased 1.1 ± 0.3 μl min−1 mmHg−1 above baseline with BIBP3226, and 2.4 ± 0.4 μl min−1 mmHg−1 above baseline with prazosin (both P < 0.05). The increase in vascular conductance during combined blockade (5.1 ± 0.7 μl min−1 mmHg−1) was greater than the sum of the independent BIBP3226 and prazosin responses (P < 0.05). In females, basal hindlimb vascular conductance was unaffected by Y1‐receptor blockade. However, α1‐receptor blockade resulted in a 3.5 ± 0.6 μl min−1 mmHg−1 increase in vascular conductance above baseline, which was not different than the combined blockade condition. Males had greater skeletal muscle neuropeptide Y concentration (P < 0.05; ELISA) than females. Furthermore, compared with females, male skeletal muscle contained greater Y1‐receptor expression (P < 0.05; Western blot). It was concluded that, under baseline conditions, agonist and receptor‐based mechanisms for Y1‐receptor dependent control of vascular conductance in skeletal muscle was greater in male versus female rats.

A Simple ''Streak Length Method'' for Quantifying and Characterizing Red Blood Cell Velocity Profiles and Blood Flow in Rat Skeletal Muscle Arterioles

Please cite this paper as: Al‐Khazraji BK, Novielli NM, Goldman D, Medeiros PJ, Jackson DN. A simple “Streak Length Method” for quantifying and characterizing red blood cell velocity profiles and blood flow in rat skeletal muscle arterioles. Microcirculation 19: 327–335, 2012.

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

The sympathetic nervous system increases skeletal muscle arteriolar resistance by activating adrenergic, peptidergic and purinergic receptors, which may depend on network topology. To date, there has been limited work conducted on topologically‐dependent sympathetic nervous system control in continuously branching skeletal muscle microvascular networks. In the present study, we investigated how arterioles respond to activation of receptors for sympathetic neurotransmitters based on their location within continuously branching arteriolar trees in skeletal muscle. For the first time, we show differential order‐dependent responses to adrenergic, peptidergic and purinergic agonists in continuously branching arteriolar trees. These results provide novel and detailed network data describing full‐range sympathetic control in the skeletal muscle microcirculation. This work provides much needed experimental data, which can be applied to mathematical models of skeletal muscle blood flow and oxygen transport.

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.

Estrogen modulates the contribution of neuropeptide Y to baseline hindlimb blood flow control in female Sprague-Dawley rats

The purpose of this study was to determine the role of estrogen in neuropeptide Y (NPY) and Y(1) receptor (Y(1)R)-mediated vascular responses in female rats. Based on earlier work from our laboratory that female rats lacked an NPY contribution to hindlimb vascular conductance relative to males, we tested the hypothesis that estrogen modulates Y(1)R-mediated hindlimb blood flow control. Thus it was expected that ovariectomy would: 1) increase skeletal muscle Y(1)R expression, 2) decrease skeletal muscle Y(2) receptor (Y(2)R) expression, 3) decrease peptidase activity, and/or 4) increase overall skeletal muscle NPY concentration. Separate groups of control (CTL), ovariectomized (OVX), and OVX + 17beta-estradiol replacement (OVX + E(2); 21-day pellet) rats were studied. Animals were anesthetized and given localized hindlimb delivery of BIBP-3226 (Y(1)R antagonist), while femoral artery blood flow and blood pressure were recorded. Tissue samples from the white and red vastus lateralis muscle were extracted to examine Y(1)R and Y(2)R expression, peptidase activity, and NPY concentration. We found that Y(1)R blockade resulted in increased baseline hindlimb blood flow and vascular conductance in OVX rats, whereas no change was noted in CTL or OVX + E(2) groups (P < 0.05). This enhanced functional effect in the OVX group aligned with greater skeletal muscle Y(1)R expression in white vastus muscle and a substantial increase in NPY concentration in both white and red vastus muscle compared with CTL and OVX + E(2) groups. There was no change in Y(2)R expression or peptidase activity among the groups. These data support the hypothesis that estrogen blunts Y(1)R activation in the rat hindlimb through an effect on Y(1)R expression and NPY concentration.

An automated cell-counting algorithm for fluorescently-stained cells in migration assays

A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting.

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.