Platelet bioenergetic screen in sickle cell patients reveals mitochondrial complex V inhibition, which contributes to platelet activation.

Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed.

Nitrite augments glucose uptake in adipocytes through the protein kinase A-dependent stimulation of mitochondrial fusion.

Though it is well accepted that adipose tissue is central in the regulation of glycemic homeostasis, the molecular mechanisms governing adipocyte glucose uptake remain unclear. Recent studies demonstrate that mitochondrial dynamics (fission and fusion) regulate lipid accumulation and differentiation in adipocytes. However, the role of mitochondrial dynamics in glucose homeostasis has not been explored.

Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion.

Aims: Nitrite (NO2(-)), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored.

Platelet mitochondrial function: from regulation of thrombosis to biomarker of disease.

Circulating blood platelets contain small numbers of fully functional mitochondria. Accumulating evidence demonstrates that these mitochondria regulate the pro-thrombotic function of platelets through not only energy generation, but also redox signalling and the initiation of apoptosis. Beyond its regulation of haemostasis, platelet mitochondrial function has also traditionally been used to identify and study mitochondrial dysfunction in human disease, owing to the easy accessibility of platelets compared with other metabolically active tissues.

Novel peptide for attenuation of hypoxia-induced pulmonary hypertension via modulation of nitric oxide release and phosphodiesterase -5 activity.

Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5'-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation.

Arginine and asymmetric dimethylarginine in puromycin aminonucleoside-induced chronic kidney disease in the rat.

Background/aims: Reduced renal L-arginine (L-Arg) synthesis/transport, induction of arginases and increased endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA) will inhibit NO production. This study investigated pathways of L-Arg synthesis/uptake/utilization, ADMA degradation and oxidant/antioxidants in puromycin aminonucleoside (PAN) chronic kidney disease (CKD).

Methods: Rats were given low- (LD) or high-dose (HD) PAN and followed for 11 weeks for proteinuria.

Hypoxic upregulation of arginase II in human lung endothelial cells.

Activated arginase has been implicated in many diseases including cancer, immune cell dysfunction, infections, and vascular disease. Enhanced arginase activity has been reported in lungs of patients with pulmonary artery hypertension. We used hypoxia as a model for pulmonary hypertension and studied the effect of exposure to hypoxia on arginase activity in human lung microvascular endothelial cells (HMVEC).

Endothelial arginase II responds to pharmacological inhibition by elevation in protein level.

Arginase is an enzyme which converts arginine to ornithine and urea. Recently, arginase has been implicated in many physiological and pathological processes including vascular diseases. Inhibition of arginase activity by pharmacological inhibitors is a useful tool to study the biology of arginases and their possible role in therapy.

Effects of angiotensin type 1 receptor blockade on arginine and ADMA synthesis and metabolic pathways in fawn-hooded hypertensive rats.

Background: The fawn-hooded hypertensive (FHH) rat develops spontaneous glomerulosclerosis that is ameliorated by inhibition of the angiotensin II type 1 receptor (AT-1). Since kidney damage is associated with nitric oxide (NO) deficiency, we investigated how AT-1 antagonism influenced nitric oxide synthase (NOS), as well as NOS substrate [L-arginine (L-Arg)] and inhibitor [asymmetric dimethylarginine (ADMA)]. L-Arg is synthesized by renal argininosuccinate synthase/argininosuccinate lyase (ASS/ASL) and then either consumed within the kidney by arginase II or NOS or released into the circulation.

Could uric acid be a modifiable risk factor in subjects with pulmonary hypertension?

A high serum uric acid is common in subjects with pulmonary hypertension. The increase in serum uric acid may be a consequence of the local tissue ischemia and/or hypoxia, and it may also result from other factors independent of ischemia or hypoxia that occur in various forms of pulmonary hypertension. While classically viewed as a secondary phenomenon, recent studies suggest that hyperuricemia may also have a role in mediating the local vasoconstriction and vascular remodeling in the pulmonary vasculature.

Induction of nitric oxide by erythropoietin is mediated by the {beta} common receptor and requires interaction with VEGF receptor 2.

Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) have profound effects on the endothelium and endothelial progenitor cells (EPCs), which originate from the bone marrow and differentiate into endothelial cells. Both EPO and VEGF have demonstrated an ability to increase the number and performance properties of EPCs. EPC behavior is highly dependent on nitric oxide (NO), and both VEGF and EPO can stimulate intracellular NO.

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells.

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC).

Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress.

Uric acid is considered a major antioxidant in human blood that may protect against aging and oxidative stress. Despite its proposed protective properties, elevated levels of uric acid are commonly associated with increased risk for cardiovascular disease and mortality. Furthermore, recent experimental studies suggest that uric acid may have a causal role in hypertension and metabolic syndrome.

Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation.

1. Myristoylated pseudosubstrate of PKCzeta (mPS) - a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCzeta pseudosubstrate region -- is considered a selective cell-permeable inhibitor of PKCzeta. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation.

A causal role for uric acid in fructose-induced metabolic syndrome.

The worldwide epidemic of metabolic syndrome correlates with an elevation in serum uric acid as well as a marked increase in total fructose intake (in the form of table sugar and high-fructose corn syrup). Fructose raises uric acid, and the latter inhibits nitric oxide bioavailability. Because insulin requires nitric oxide to stimulate glucose uptake, we hypothesized that fructose-induced hyperuricemia may have a pathogenic role in metabolic syndrome.

Hyperuricemia induces endothelial dysfunction.

Background: Hyperuricemia has been linked to cardiovascular and renal diseases, possibly through the generation of reactive oxygen species (ROS) and subsequent endothelial dysfunction. The enzymatic effect of xanthine oxidase is the production of ROS and uric acid. Studies have shown that inhibiting xanthine oxidase with allopurinol can reverse endothelial dysfunction.

Molecular cloning, characterization, and chromosomal assignment of porcine cationic amino acid transporter-1.

We have cloned and characterized the gene encoding the porcine cationic amino acid transporter, member 1 (CAT-1) (HGMW-approved gene symbol SLC7A1) from porcine pulmonary artery endothelial cells. The porcine SLC7A1 encodes 629 deduced amino acid residues showing a higher degree of sequence similarity with the human counterpart (91.1%) than with the rat (87.

Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-alpha activity.

Pertussis toxin (PTX) induces activation of l-arginine transport in pulmonary artery endothelial cells (PAEC). The effects of PTX on l-arginine transport appeared after 6 h of treatment and reached maximal values after treatment for 12 h. PTX-induced changes in l-arginine transport were not accompanied by changes in expression of cationic amino acid transporter (CAT)-1 protein, the main l-arginine transporter in PAEC.

Metabolism of dynorphins by peptidases of pulmonary artery endothelial cells.

Degradation of several dynorphins by peptidases expressed in cultured porcine pulmonary artery endothelial cells was studied by incubation of the peptide in cell suspensions followed by electrospray ionization and tandem mass spectrometric analyses. Under the in vitro conditions applied, only the metabolism of dynorphin A1-8 occurred in a significant extent. Studies involving specific peptidase inhibitors indicated that mainly bestatin-sensitive aminopeptidases, thiorphan-sensitive endopeptidases, and cFPAAF-pAB-sensitive endopeptidases expressed by the endothelial cells were involved in the process that converted dynorphin A1-8 to dynorphin A2-8, dynorphin A1-6, and leucine enkephalin (dynorphin A1-5), respectively.

Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells.

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediated l-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter.