Cynthia J. Meininger

Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH(4)), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH(4) levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH(4) levels with sepiapterin increased NO production, suggesting that BH(4) deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH(4). GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

Effects of ageing and exercise training on endothelium‐dependent vasodilatation and structure of rat skeletal muscle arterioles

Ageing reduces endothelium‐dependent vasodilatation in humans and animals, and in humans, exercise training reverses the ageing‐associated reduction in endothelium‐dependent vasodilatation. The purpose of this study was to determine the mechanism(s) by which 10–12 weeks of treadmill exercise enhances endothelium‐dependent vasodilatation in muscles of differing fibre composition from young and old rats. Three‐ and 22‐month‐old male Fischer 344 rats were assigned to young sedentary, young exercise‐trained, old sedentary, or old exercise‐trained groups. Arterioles were isolated from the soleus and gastrocnemius muscles; luminal diameter changes were determined in response to the endothelium‐dependent vasodilator acetylcholine (ACh, 10−9–10−4 mol l−1) alone and in the presence of the nitric oxide synthase (NOS) inhibitor l‐NAME (10−5 mol l−1) or the combination of l‐NAME and the cyclooxygenase inhibitor indomethacin (10−5 mol l−1). Training ameliorated the ageing‐induced reduction in endothelium‐dependent vasodilatation in soleus muscle arterioles. Treatment with l‐NAME alone and in combination with indomethacin abolished differences in ACh vasodilatation occurring with ageing and training. Expression of endothelial NOS (eNOS) mRNA in soleus arterioles was unaltered by ageing, whereas eNOS protein was increased with age; training elevated both eNOS mRNA and protein. In gastrocnemius muscle arterioles, ageing did not alter maximal vasodilatation, but ageing and training increased maximal arteriolar diameter. These results demonstrate that ageing‐induced reductions and training‐induced enhancement of endothelial vasodilatation both occur through the nitric oxide signalling mechanism in highly oxidative skeletal muscle, but ageing and training do not appear to act on the same portion of the signalling cascade.

Arginine nutrition in development, health and disease.

Abstract As a precursor of nitric oxide, polyamines and other molecules with enormous biologic importance, L‐arginine plays versatile key roles in nutrition and metabolism. Arginine is an essential amino acid in the fetus and neonate, and is conditionally an essential nutrient for adults, particularly in certain disease conditions. L‐Arginine administration is beneficial in improving reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver and immune functions, and in facilitating wound healing. The effect of L‐arginine in treating many common health problems is unique among amino acids, and offers great promise for improved health and well‐being in the future.

γ-Aminobutyric Acid Inhibits Cholangiocarcinoma Growth by Cyclic AMP–Dependent Regulation of the Protein Kinase A/Extracellular Signal-Regulated Kinase 1/2 Pathway

We studied the effect of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in the regulation of cholangiocarcinoma growth. We determined the in vitro effect of GABA on the proliferation of the cholangiocarcinoma cell lines (Mz-ChA-1, HuH-28, and TFK-1) and evaluated the intracellular pathways involved. The effect of GABA on migration of Mz-ChA-1 cells was also evaluated. In vivo, Mz-ChA-1 cells were s.c. injected in athymic mice, and the effects of GABA on tumor size, tumor cell proliferation, apoptosis, collagen quantity, and the expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-C (cancer growth regulators) were measured after 82 days. GABA decreased in vitro cholangiocarcinoma growth in a time-dependent and dose-dependent manner, by both cyclic AMP/protein kinase A- and D-myo-inositol-1,4,5-thriphosphate/Ca(2+)-dependent pathways, leading to down-regulation of extracellular signal-regulated kinase 1/2 phosphorylation. Blocking of GABA(A), GABA(B), and GABA(C) receptors prevented GABA inhibition of cholangiocarcinoma proliferation. GABA inhibited Mz-ChA-1 cell migration and, in vivo, significantly decreased tumor volume, tumor cell proliferation, and VEGF-A/C expression whereas increasing apoptosis compared with controls. An increase in collagen was evident in GABA-treated tumors. GABA decreases biliary cancer proliferation and reduces the metastatic potential of cholangiocarcinoma. GABA may represent a therapeutic agent for patients affected by malignancies of the biliary tract.

Nitric oxide and energy metabolism in mammals

Nitric oxide (NO) is a signaling molecule synthesized from L‐arginine by NO synthase in animals. Increasing evidence shows that NO regulates the mammalian metabolism of energy substrates and that these effects of NO critically depend on its concentrations at the reaction site and the period of exposure. High concentrations of NO (in the micromolar range) irreversibly inhibit complexes I, II, III, IV, and V in the mitochondrial respiratory chain, whereas physiological levels of NO (in the nanomolar range) reversibly reduce cytochomrome oxidase. Thus, NO reduces oxygen consumption by isolated mitochondria to various extents. In intact cells, through cGMP and AMP‐activated protein kinase signaling, physiological levels of NO acutely stimulate uptake and oxidation of glucose and fatty acids by skeletal muscle, heart, liver, and adipose tissue, while inhibiting the synthesis of glucose, glycogen and fat in the insulin‐sensitive tissues, and enhancing lipolysis in white adipocytes. Chronic effects of physiological levels of NO in vivo include stimulation of angiogenesis, blood flow, mitochondrial biogenesis, and brown adipocyte development. Modulation of NO‐mediated pathways through dietary supplementation with L‐arginine or its precursor L‐citrulline may provide an effective, practical strategy to prevent and treat metabolic syndrome, including obesity, diabetes, and dyslipidemia in mammals, including humans.

Inhibition of histidine decarboxylase ablates the autocrine tumorigenic effects of histamine in human cholangiocarcinoma

Background In several tumours the endogenous activity of histidine decarboxylase (HDC), the enzyme stimulating histamine synthesis, sustains the autocrine trophic effect of histamine on cancer progression. Cholangiocarcinoma is a biliary cancer with limited treatment options. Histamine interacts with four G-protein coupled receptors, H1–H4 histamine receptors (HRs). Objective To determine the effects of histamine stimulation and inhibition of histamine synthesis (by modulation of HDC) on cholangiocarcinoma growth. Methods In vitro studies were performed using multiple human cholangiocarcinoma lines. The expression levels of the histamine synthetic machinery and HRs were evaluated along with the effects of histamine stimulation and inhibition on cholangiocarcinoma proliferation. A xenograft tumour model was used to measure tumour volume after treatment with histamine or inhibition of histamine synthesis by manipulation of HDC. Vascular endothelial growth factor (VEGF) expression was measured in cholangiocarcinoma cells concomitant with the evaluation of the expression of CD31 in endothelial cells in the tumour microenvironment. Results Cholangiocarcinoma cells display (1) enhanced HDC and decreased monoamine oxidase B expression resulting in increased histamine secretion; and (2) increased expression of H1–H4 HRs. Inhibition of HDC and antagonising H1HR decreased histamine secretion in Mz-ChA-1 cells. Long-term treatment with histamine increased proliferation and VEGF expression in cholangiocarcinoma that was blocked by HDC inhibitor and the H1HR antagonist. In nude mice, histamine increased tumour growth (up to 25%) and VEGF expression whereas inhibition of histamine synthesis (by reduction of HDC) ablated the autocrine stimulation of histamine on tumour growth (∼80%) and VEGF expression. No changes in angiogenesis (evaluated by changes in CD31 immunoreactivity) were detected in the in vivo treatment groups. Conclusion The novel concept that an autocrine loop (consisting of enhanced histamine synthesis by HDC) sustains cholangiocarcinoma growth is proposed. Drug targeting of HDC may be important for treatment of patients with cholangiocarcinoma.

Presence of glutamine:fructose-6-phosphate amidotransferase for glucosamine-6-phosphate synthesis in endothelial cells: effects of hyperglycaemia and glutamine.

Aims/hypothesis. Recent studies show that glucosamine infusion impairs endothelium-dependent blood flow in normoglycaemic rats. The pathophysiological relevance of this finding, however, depends on whether de novo glucosamine synthesis occurs in endothelial cells. The aim of this study was to test the hypothesis of whether glutamine:fructose-6-phosphate amidotransferase (the first and key regulatory enzyme in hexosamine synthesis) is present for endothelial glucosamine synthesis. Methods. Bovine venular, bovine aortic, human microvascular, human umbilical vein, and rat coronary microvascular endothelial cells were used to measure glutamine:fructose-6-phosphate amidotransferase activity. To determine glucosamine-6-phosphate synthesis in intact cells, they were incubated for 1 h in Krebs bicarbonate buffer containing 5, 15 or 30 mmol/l [U-14C]glucose and 0.5, 2 or 4 mmol/l glutamine. The [14C]Glucosamine-6-phosphate and its end products ([14C]UDP-N-acetylglucosamine and [14C]UDP-N-acetylgalactosamine) were separated by HPLC. Results. There were high glutamine:fructose-6-phosphate amidotransferase activities in all endothelial cells studied. Exposure of cells to 15 to 30 mmol/l glucose or 2 to 4 mmol/l glutamine increased enzyme activity. Glucosamine-6-phosphate, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine syntheses increased with increasing extracellular concentrations of glucose from 5 to 30 mmol/l or of glutamine from 0.5 to 4 mmol/l. Conclusion/interpretation. Our results show the presence of glutamine:fructose-6-phosphate amidotransferase for de novo glucosamine synthesis in endothelial cells and the modulation of this pathway by hyperglycaemia and glutamine. As glucosamine inhibits endothelial nitric oxide synthesis, these findings could have important implications for impaired endothelium-dependent relaxation and vascular dysfunction in diabetes mellitus. [Diabetologia (2001) 44: 196–202]

L-arginine, tetrahydrobiopterin, nitric oxide and diabetes.

Purpose of reviewThe endothelial isoform of nitric oxide synthase (eNOS) is constitutively expressed but dynamically regulated by a number of factors. Building our knowledge of this regulation is necessary to understand and modulate the bioavailability of nitric oxide, central to the cardiovascular complications of diabetes and other diseases. This review will focus on the eNOS substrate (L-arginine), its cofactor (tetrahydrobiopterin), and mechanisms related to the uncoupling of eNOS activity. Recent findingsThe global arginine bioavailability ratio has been proposed as a biomarker reflective of L-arginine availability, arginase activity, and citrulline cycling, as all of these processes impact eNOS activity. The failure of oral supplementation of tetrahydrobiopterin to recouple eNOS has emphasized the importance of the tetrahydrobiopterin to dihydrobiopterin ratio. Identification of transporters for biopterin species as well as signals that regulate endogenous arginine production have provided insight for alternative strategies to raise endothelial tetrahydrobiopterin levels while reducing dihydrobiopterin and alter eNOS activity. Finally, new information about redox regulation of eNOS itself may point to ways of controlling oxidative stress in the vasculature. SummaryRestoring proper eNOS activity is key to ameliorating or preventing cardiovascular complications of diabetes. Continued investigation is needed to uncover new means for maintaining endothelial nitric oxide bioavailability.

Visualizing the function and fate of neutrophils in sterile injury and repair

Imaging the unforeseen fate of neutrophils Inflammation that results from insults such as ischemia and reperfusion or trauma in the absence of microorganisms is known as “sterile inflammation.” Neutrophils are recruited in vast numbers during sterile inflammation and have been thought to play a detrimental role. Wang et al. used intravital microscopy to show that neutrophils actually perform helpful tasks such as removing and regenerating thermally damaged blood vessels in the liver (see the Perspective by Garner and de Visser). Moreover, neutrophils neither die nor are phagocytosed. Instead, they return to the circulation in a process called “reverse transmigration,” making a pit stop in the lungs, before ending their lives where they began—in the bone marrow. Thus, a reconsideration of the use of anti-neutrophil therapies after injury may be warranted. Science, this issue p. 111; see also p. 42 Neutrophils perform important wound-healing functions in thermally damaged tissue before returning to the bone marrow. Neutrophils have been implicated as harmful cells in a variety of inappropriate inflammatory conditions where they injure the host, leading to the death of the neutrophils and their subsequent phagocytosis by monocytes and macrophages. Here we show that in a fully repairing sterile thermal hepatic injury, neutrophils also penetrate the injury site and perform the critical tasks of dismantling injured vessels and creating channels for new vascular regrowth. Upon completion of these tasks, they neither die at the injury site nor are phagocytosed. Instead, many of these neutrophils reenter the vasculature and have a preprogrammed journey that entails a sojourn in the lungs to up-regulate CXCR4 (C-X-C motif chemokine receptor 4) before entering the bone marrow, where they undergo apoptosis.

Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels

The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium‐derived histamine relaxes MLVs.