Ghrelin is a 28-amino acid peptide predominantly produced in the stomach and secreted into the circulation. Ghrelin is found in plasma and tissues in two major forms of n-octanoyl-modified at its N-terminal third serine residue and des-acyl ghrelin. The n-octanoyl group of ghrelin is essential for its growth hormone (GH)-releasing activity and appetite regulation mediated through growth hormone secretagogue receptor (GHS-R). We demonstrated that both ghrelin and des-acyl ghrelin evoke vasodilatation at remarkably low concentrations compared to acetylcholine (ACh) in phenylephrine (PE)-constricted perfused rat mesenteric vascular bed (MVB). This was abolished in endothelium-denuded preparations and in endothelium-intact preparations exposed to either a calcium-activated potassium channel (KCa) blocker or a depolarizing stimulus. While KATP channel blockade, nitric oxide synthase and cyclooxygenase inhibition had no effect, the responses were abolished in the presence of combinations of apamin and charybdotoxin, apamin and TRAM-34, and ouabain and Ba2+. The GHS-R antagonist, [D-Lys3]-GHRP-6, per se evoked vasodilatation. Inclusion of L-756867, a peptide antagonist of classical GHS-R, failed to evoke any vasodilator response or to affect vasodilatation evoked by ghrelin. Both non-peptide agonists of GHS-R, L-166446 and L-163255, demonstrated concentration-dependent decreases in perfusion pressure. All short peptides encompassing the first 20, 16, 10, 6, 4, and 3 residues of des-acyl ghrelin were able to evoke vasodilator responses to the same extent as des-acyl ghrelin. However, vasodilatation to single amino acids, L-serine and glycine, were significantly attenuated. Streptozotocin (STZ)-induced diabetes increased plasma ghrelin concentration. Diabetes for 4-weeks did not cause any significant reduction in ghrelin-evoked vasodilatation, whereas 8-weeks diabetes significantly reduced ghrelin-evoked vasodilatation. In contrast to ghrelin, there was a duration-dependent fall in vasodilator response to ACh from 4- to 8-weeks diabetes. These data suggest that the vasodilatation evoked by ghrelin is mediated by endothelium-dependent hyperpolarization (EDHF) by mechanism(s) that are independent of classical GHS-R activation. In addition, EDHF-dependent ghrelin-evoked vasodilator responses may not be affected, at least in the early stages of STZ diabetes, whereas the responses to ACh, predominantly mediated through nitric oxide, are progressively diminished right from the early stages of endothelial dysfunction in STZ diabetic rats.

Ghrelin is a 28-amino acid peptide predominantly produced in the stomach and secreted into the circulation. Ghrelin is found in plasma and tissues in two major forms of n-octanoyl-modified at its N-terminal third serine residue and des-acyl ghrelin. The n-octanoyl group of ghrelin is essential for its growth hormone (GH)-releasing activity and appetite regulation mediated through growth hormone secretagogue receptor (GHS-R). We demonstrated that both ghrelin and des-acyl ghrelin evoke vasodilatation at remarkably low concentrations compared to acetylcholine (ACh) in phenylephrine (PE)-constricted perfused rat mesenteric vascular bed (MVB). This was abolished in endothelium-denuded preparations and in endothelium-intact preparations exposed to either a calcium-activated potassium channel (KCa) blocker or a depolarizing stimulus. While KATP channel blockade, nitric oxide synthase and cyclooxygenase inhibition had no effect, the responses were abolished in the presence of combinations of apamin and charybdotoxin, apamin and TRAM-34, and ouabain and Ba2+. The GHS-R antagonist, [D-Lys3]-GHRP-6, per se evoked vasodilatation. Inclusion of L-756867, a peptide antagonist of classical GHS-R, failed to evoke any vasodilator response or to affect vasodilatation evoked by ghrelin. Both non-peptide agonists of GHS-R, L-166446 and L-163255, demonstrated concentration-dependent decreases in perfusion pressure. All short peptides encompassing the first 20, 16, 10, 6, 4, and 3 residues of des-acyl ghrelin were able to evoke vasodilator responses to the same extent as des-acyl ghrelin. However, vasodilatation to single amino acids, L-serine and glycine, were significantly attenuated. Streptozotocin (STZ)-induced diabetes increased plasma ghrelin concentration. Diabetes for 4-weeks did not cause any significant reduction in ghrelin-evoked vasodilatation, whereas 8-weeks diabetes significantly reduced ghrelin-evoked vasodilatation. In contrast to ghrelin, there was a duration-dependent fall in vasodilator response to ACh from 4- to 8-weeks diabetes. These data suggest that the vasodilatation evoked by ghrelin is mediated by endothelium-dependent hyperpolarization (EDHF) by mechanism(s) that are independent of classical GHS-R activation. In addition, EDHF-dependent ghrelin-evoked vasodilator responses may not be affected, at least in the early stages of STZ diabetes, whereas the responses to ACh, predominantly mediated through nitric oxide, are progressively diminished right from the early stages of endothelial dysfunction in STZ diabetic rats.

No description available.

Vascular dysfunction is linked with increased free radical generation and is a major contributor to the high mortality rates observed in diabetes. Several probable sources of free radical generation have been suggested in diabetes, including cytochrome P450 (CYP) monooxygenase-dependent pathways. CYP-mediated superoxide production reduces nitric oxide (NO) bioavailability. In this study, we focus on the contribution of CYP monooxygenase enzyme-generated reactive oxygen species in vascular dysfunction in an experimental model of type II diabetes mellitus. The purpose of this study is to test the hypothesis that sulfaphenazole treatment can restore diabetic endothelial function in db/db mice. Diabetic male mice (db/db strain) and their age-matched controls received daily intraperitoneal injections of either the CYP 2C inhibitor sulfaphenazole (5 mg/kg) or saline (vehicle control) for 8 weeks. Fasting plasma glucose levels were measured before starting, during, and after finishing the treatment. As well, plasma levels of 8-isoprostane (as a marker of oxidative stress) and nitrite levels of aortic tissue (as a marker of NO bioavailability) were determined. Although sulfaphenazole did not change endothelium-dependent vasodilation in WT mice, it restored endothelial-mediated relaxation in treated db/db mice. We concluded that CYP 2C inhibition by sulfaphenazole reduces oxidative stress (measured as plasma levels of 8-isoprostane), increases NO bioavailability (measured as NOj) and restores endothelial function in db/db mice without affecting plasma glucose levels. Based on our findings, we speculate that inhibition of free radical generating CYP monooxygenase enzymes restores endothelium-dependent vasodilation induced by acetylcholine. In addition, it reduces oxidative stress and increases NO bioavailability.

Vascular dysfunction is linked with increased free radical generation and is a major contributor to the high mortality rates observed in diabetes. Several probable sources of free radical generation have been suggested in diabetes, including cytochrome P450 (CYP) monooxygenase-dependent pathways. CYP-mediated superoxide production reduces nitric oxide (NO) bioavailability. In this study, we focus on the contribution of CYP monooxygenase enzyme-generated reactive oxygen species in vascular dysfunction in an experimental model of type II diabetes mellitus. The purpose of this study is to test the hypothesis that sulfaphenazole treatment can restore diabetic endothelial function in db/db mice. Diabetic male mice (db/db strain) and their age-matched controls received daily intraperitoneal injections of either the CYP 2C inhibitor sulfaphenazole (5 mg/kg) or saline (vehicle control) for 8 weeks. Fasting plasma glucose levels were measured before starting, during, and after finishing the treatment. As well, plasma levels of 8-isoprostane (as a marker of oxidative stress) and nitrite levels of aortic tissue (as a marker of NO bioavailability) were determined. Although sulfaphenazole did not change endothelium-dependent vasodilation in WT mice, it restored endothelial-mediated relaxation in treated db/db mice. We concluded that CYP 2C inhibition by sulfaphenazole reduces oxidative stress (measured as plasma levels of 8-isoprostane), increases NO bioavailability (measured as NOj) and restores endothelial function in db/db mice without affecting plasma glucose levels. Based on our findings, we speculate that inhibition of free radical generating CYP monooxygenase enzymes restores endothelium-dependent vasodilation induced by acetylcholine. In addition, it reduces oxidative stress and increases NO bioavailability.

Submucosal arterioles in the small intestine are the main point of control for gastrointestinal (GI) circulation as they are the final resistance vessels feeding the highly perfused mucosal layer. Ischemia can lead to pathophysiology of a variety of GI tissues. In chronic intestinal inflammation, alterations in blood flow have been purported to be involved in disease etiology. The aim of this study was to characterize purinergic neurotransmitter pathways involved in physiological submucosal arteriole diameter control by the enteric nervous system long vasodilatory reflex (LVD) and to establish a protocol to determine how inflammatory neural changes affect vasodilation in the small intestine. Following euthanasia, segments of small intestine were harvested from adult male guinea pigs and changes in nerve stimulated small intestine submucosal arteriole diameter were identified using videomicroscopy techniques; vessels were preconstricted and nicotinic cholinergic transmission was blocked with hexamethonium. Purinergic receptor antagonists were applied. Immunohistochemical analysis was conducted to identify P2Y1 receptors localization. In a subset of experiments sensory neuronal excitability was initiated using phorbol dibutyrate (PDBu) shown previously to induce hyperexcitability in the sensory neurons similar to changes found in intestinal inflammation. In these experiments, intestinal segments were placed into a novel dual chamber bath separated into two portions and PDBu was applied unilaterally. Blood vessel vasodilation was either abolished or decreased by both suramin (100 μM, n=6), a non-specific P2 purinergic antagonist. MRS 2179 (10 μM, n=5), a P2Y1 specific antagonist, also decreased vasodilation, which suggests that there is a purinergic neurotransmission component to the LVD mediated by P2 receptors, including the P2Y1 subtype. Immunohistochemistry identified P2Y1 receptor staining that was uniformly punctated in both the myenteric and submucosal plexuses but specific neuronal locations of the receptor could not be identified. Nerve stimulated vasodilation was not altered by application of PDBu suggesting that neuronal hypersensitivity did not modify vessel dilation. Taken together these data suggest that purinergic receptor pathways contribute to the LVD reflex under normal conditions however more experiments are still required to fully elucidate how these pathways are affected /altered by intestinal inflammation. / Thesis (Master, Physiology) -- Queen's University, 2012-05-30 11:38:43.324

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Vascular dysfunction is linked with increased free radical generation and is a major contributor to the high mortality rates observed in diabetes. Several probable sources of free radical generation have been suggested in diabetes, including cytochrome P450 (CYP) monooxygenase-dependent pathways. CYP-mediated superoxide production reduces nitric oxide (NO) bioavailability. In this study, we focus on the contribution of CYP monooxygenase enzyme-generated reactive oxygen species in vascular dysfunction in an experimental model of type II diabetes mellitus. The purpose of this study is to test the hypothesis that sulfaphenazole treatment can restore diabetic endothelial function in db/db mice. Diabetic male mice (db/db strain) and their age-matched controls received daily intraperitoneal injections of either the CYP 2C inhibitor sulfaphenazole (5 mg/kg) or saline (vehicle control) for 8 weeks. Fasting plasma glucose levels were measured before starting, during, and after finishing the treatment. As well, plasma levels of 8-isoprostane (as a marker of oxidative stress) and nitrite levels of aortic tissue (as a marker of NO bioavailability) were determined. Although sulfaphenazole did not change endothelium-dependent vasodilation in WT mice, it restored endothelial-mediated relaxation in treated db/db mice. We concluded that CYP 2C inhibition by sulfaphenazole reduces oxidative stress (measured as plasma levels of 8-isoprostane), increases NO bioavailability (measured as NOj) and restores endothelial function in db/db mice without affecting plasma glucose levels. Based on our findings, we speculate that inhibition of free radical generating CYP monooxygenase enzymes restores endothelium-dependent vasodilation induced by acetylcholine. In addition, it reduces oxidative stress and increases NO bioavailability. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Aging is associated with attenuated blood flow and vasodilator responses during rhythmic exercise. Older adults also demonstrate attenuated blood flow and vasodilator responses following single skeletal muscle contractions (contraction-induced rapid onset vasodilation, ROV) within the forearm. These age-associated attenuations within the forearm have been demonstrated to be a result of endothelial and neural mechanisms. The objective of this research was to examine: 1) whether age-associated attenuations within the forearm are from mechanical factors; 2) whether age-associated attentions in ROV are present within the leg, as well as explore potential mechanisms for these age-associated attenuations in ROV; 3) examine whether aging is associated with a slower rate of adjustment in vasodilation (vasodilator kinetics) during rhythmic exercise preceding steady-state exercise; and 4) examine approaches to ameliorate age-related attenuations in blood flow and vasodilation within the leg across the entire exercise transient (onset to steady-state). The novel findings of this research are that 1) age-associated attenuations in ROV within the forearm are independent of mechanical factors; 2) older adults demonstrate attenuated ROV responses within the leg; 3) age-related attenuations in ROV within the leg are not explained by enhanced sympathetic adrenergic vasoconstriction; 4) older adults exhibit prolonged vasodilator kinetics preceding steady-state exercise; and 5) when examined in a cross-sectional design chronic exercise training improves ROV, vasodilator kinetics, as well as steady-state blood flow and vasodilator responses in older adults; 6) acute supplementation with dietary nitrate fails to exert any effect on blood flow and vasodilator responses during any domain of exercise. Collectively, this work establishes that aging is associated with reductions in blood flow and vasodilation across the entire exercise transient (onset to steady-state) within the leg, which is offset by chronic exercise training. Mechanistically, the current data suggests that mechanical and sympathetic factors do not explain age-related reductions in ROV in the arm and leg, respectively. Furthermore, acute supplementation of dietary nitrate does not impact leg blood flow and vasodilator responses in older adults during any domain of the exercise transient.

Control of blood flow to skeletal muscle is essential to maintain the overall homeostasis of an organism. The primary route that skeletal muscle uses to accommodate an increased metabolic demand associated with physical activity is to increase its blood flow through functional hyperemia. The importance of functional hyperemia in ensuring proper skeletal muscle function spurred 130 years of investigation into the mechanism(s) regulating its occurrence.Despite not identifying the essential factor(s) for controlling skeletal muscle blood flow, the last century of investigation has uncovered much about the process; including the observation that skeletal muscle functional hyperemia is impaired with ischemic disease. In patients, this can result in immobility, chronic ulcerations, gangrene, and at worst, amputation. To develop efficacious therapies, we as scientists must develop a better understanding of the molecular mechanisms underlying impaired vascular function during ischemia.The goal of this work was to lay the foundation for investigations examining the role of specific gene products involved in modulating blood flow control during ischemic revascularization by assessing vascular function in the mouse following an ischemic event. Unique among research animals, the mouse is routinely accessible for targeted genetic disruption, which allows investigators to assess the requirement of specific gene-products in a physiological process. Unfortunately, to date, no publication that I am aware of describes blood flow measurement to contracting mouse skeletal muscle following an ischemic/revascularization event. Therefore, the primary objective of this work was to assess vascular function in genetically unaltered animals.I found that unlike other species thus far examined, vascular dysfunction is not an obligatory response to hindlimb ischemic revascularization in the mouse. Ex vivo vasodilation responses to acetylcholine were statistically significantly impaired in the muscular branch artery 14 days following an ischemic event. However, using a newly developed fluorescent microsphere-based approach for determining skeletal muscle blood flow, I found that functional hyperemia was similar for the gracilis posterior muscle between non-ischemic and day-14 ischemic animals. In light of the primary literature, these findings suggest that vascular growth, and not ischemia per se is the primary regulator of vascular function during health and disease.