-
Journal of Bacteriology Apr 2017Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central...
Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including and , the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in PAO1. First, results revealed that can utilize , -dimethyl-l-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the gene was observed to be induced in the presence of methylarginines, including -monomethyl-l-arginine (l-NMMA) and ADMA. Third, induction of the gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for imethylarginine imethylminohydrolase egulator. Interestingly, DdaR and its target gene are conserved only among strains, suggesting that this particular species has evolved to utilize methylarginines from its environment. Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among strains.
Topics: Amidohydrolases; Arginine; Bacterial Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Pseudomonas aeruginosa; RNA Polymerase Sigma 54; omega-N-Methylarginine
PubMed: 28167521
DOI: 10.1128/JB.00001-17 -
Journal of Applied Physiology... Dec 2017Broxterman RM, Trinity JD, Gifford JR, Kwon OS, Kithas AC, Hydren JR, Nelson AD, Morgan DE, Jessop JE, Bledsoe AD, Richardson RS. Single passive leg movement assessment...
Broxterman RM, Trinity JD, Gifford JR, Kwon OS, Kithas AC, Hydren JR, Nelson AD, Morgan DE, Jessop JE, Bledsoe AD, Richardson RS. Single passive leg movement assessment of vascular function: contribution of nitric oxide. J Appl Physiol 123: 1468-1476, 2017. First published August 31, 2017; doi:10.1152/japplphysiol.00533.2017.-The assessment of passive leg movement (PLM)-induced leg blood flow (LBF) and vascular conductance (LVC) is a novel approach to assess vascular function that has recently been simplified to only a single PLM (sPLM), thereby increasing the clinical utility of this technique. As the physiological mechanisms mediating the robust increase in LBF and LVC with sPLM are unknown, we tested the hypothesis that nitric oxide (NO) is a major contributor to the sPLM-induced LBF and LVC response. In nine healthy men, sPLM was performed with and without NO synthase inhibition by intra-arterial infusion of N-monomethyl-l-arginine (l-NMMA). Doppler ultrasound and femoral arterial pressure were used to determine LBF and LVC, which were characterized by the peak change (ΔLBF and ΔLVC) and area under the curve (LBF and LVC). l-NMMA significantly attenuated ΔLBF [492 ± 153 (l-NMMA) vs. 719 ± 238 (control) ml/min], LBF [57 ± 34 (l NMMA) vs. 147 ± 63 (control) ml], ΔLVC [4.7 ± 1.1 (l-NMMA) vs. 8.0 ± 3.0 (control) ml·min·mmHg], and LVC [0.5 ± 0.3 (l-NMMA) vs. 1.6 ± 0.9 (control) ml/mmHg]. The magnitude of the NO contribution to LBF and LVC was significantly correlated with the magnitude of the control responses ( r = 0.94 for ΔLBF, r = 0.85 for LBF, r = 0.94 for ΔLVC, and r = 0.95 for LVC). These data establish that the sPLM-induced hyperemic and vasodilatory response is predominantly (~65%) NO-mediated. As such, sPLM appears to be a promising, simple, in vivo assessment of NO-mediated vascular function and NO bioavailability. NEW & NOTEWORTHY Passive leg movement (PLM), a novel assessment of vascular function, has been simplified to a single PLM (sPLM), thereby increasing the clinical utility of this technique. However, the role of nitric oxide (NO) in mediating the robust sPLM hemodynamic responses is unknown. This study revealed that sPLM induces a hyperemic and vasodilatory response that is predominantly NO-mediated and, as such, appears to be a promising simple, in vivo, clinical assessment of NO-mediated vascular function and, therefore, NO bioavailability.
Topics: Adult; Arterial Pressure; Enzyme Inhibitors; Hemodynamics; Humans; Hyperemia; Leg; Male; Movement; Nitric Oxide; Regional Blood Flow; Vasodilation; Young Adult; omega-N-Methylarginine
PubMed: 28860173
DOI: 10.1152/japplphysiol.00533.2017 -
American Journal of Physiology. Heart... Mar 2016We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a...
We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a greater contribution of nitric oxide synthase and cyclooxygenase to β-adrenergic-mediated vasodilation compared with men. Forearm blood flow (Doppler ultrasound) was measured in young men (n = 29, 26 ± 1 yr) and women (n = 33, 25 ± 1 yr) during intra-arterial infusion of isoproterenol (β-adrenergic agonist). In subset of subjects, isoproterenol responses were examined before and after local inhibition of nitric oxide synthase [N(G)-monomethyl-l-arginine (l-NMMA); 6 male/10 female] and/or cyclooxygenase (ketorolac; 5 male/5 female). Vascular conductance (blood flow ÷ mean arterial pressure) was calculated to assess vasodilation. Vascular conductance increased with isoproterenol infusion (P < 0.01), and this effect was not different between men and women (P = 0.41). l-NMMA infusion had no effect on isoproterenol-mediated dilation in men (P > 0.99) or women (P = 0.21). In contrast, ketorolac infusion markedly increased isoproterenol-mediated responses in both men (P < 0.01) and women (P = 0.04) and this rise was lost with subsequent l-NMMA infusion (men, P < 0.01; women, P < 0.05). β-Adrenergic vasodilation is not different between men and women and sex differences in the independent contribution of nitric oxide synthase and cyclooxygenase to β-mediated vasodilation are not present. However, these data are the first to demonstrate β-adrenoceptor activation of cyclooxygenase suppresses nitric oxide synthase signaling in human forearm microcirculation and may have important implications for neurovascular control in both health and disease.
Topics: Adrenergic beta-Agonists; Adult; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Female; Forearm; Humans; Infusions, Intra-Arterial; Isoproterenol; Ketorolac; Male; Microcirculation; Nitric Oxide Synthase; Prostaglandin-Endoperoxide Synthases; Sex Factors; Ultrasonography, Doppler; Vasodilation; omega-N-Methylarginine
PubMed: 26747505
DOI: 10.1152/ajpheart.00886.2015 -
American Journal of Physiology. Heart... Mar 2016The proposed mechanistic link between the age-related attenuation in vascular function and free radicals is an attractive hypothesis; however, direct evidence of free...
The proposed mechanistic link between the age-related attenuation in vascular function and free radicals is an attractive hypothesis; however, direct evidence of free radical attenuation and a concomitant improvement in vascular function in the elderly is lacking. Therefore, this study sought to test the hypothesis that ascorbic acid (AA), administered intra-arterially during progressive handgrip exercise, improves brachial artery (BA) vasodilation in a nitric oxide (NO)-dependent manner, by mitigating free radical production. BA vasodilation (Doppler ultrasound) and free radical outflow [electron paramagnetic resonance (EPR) spectroscopy] were measured in seven healthy older adults (69 ± 2 yr) during handgrip exercise at 3, 6, 9, and 12 kg (∼13-52% of maximal voluntary contraction) during the control condition and nitric oxide synthase (NOS) inhibition via N(G)-monomethyl-L-arginine (L-NMMA), AA, and coinfusion of l-NMMA + AA. Baseline BA diameter was not altered by any of the treatments, while L-NMMA and L-NMMA + AA diminished baseline BA blood flow and shear rate. AA improved BA dilation compared with control at 9 kg (control: 6.5 ± 2.2%, AA: 10.9 ± 2.5%, P = 0.01) and 12 kg (control: 9.5 ± 2.7%, AA: 15.9 ± 3.7%, P < 0.01). NOS inhibition blunted BA vasodilation compared with control and when combined with AA eliminated the AA-induced improvement in BA vasodilation. Free radical outflow increased with exercise intensity but, interestingly, was not attenuated by AA. Collectively, these results indicate that AA improves BA vasodilation in the elderly during handgrip exercise through an NO-dependent mechanism; however, this improvement appears not to be the direct consequence of attenuated free radical outflow from the forearm.
Topics: Aged; Ascorbic Acid; Brachial Artery; Enzyme Inhibitors; Exercise; Female; Free Radicals; Hand Strength; Humans; Infusions, Intra-Arterial; Male; Nitric Oxide; Nitric Oxide Synthase; Regional Blood Flow; Vasodilation; omega-N-Methylarginine
PubMed: 26801312
DOI: 10.1152/ajpheart.00817.2015 -
American Journal of Physiology. Heart... Jan 2022Central adiposity is associated with greater sympathetic support of blood pressure. β-adrenergic receptors (β-AR) buffer sympathetically mediated vasoconstriction and...
Central adiposity is associated with greater sympathetic support of blood pressure. β-adrenergic receptors (β-AR) buffer sympathetically mediated vasoconstriction and β-AR-mediated vasodilation is attenuated in preclinical models of obesity. With this information, we hypothesized β-AR vasodilation would be lower in obese compared with normal weight adults. Because β-AR vasodilation in normal weight adults is limited by cyclooxygenase (COX) restraint of nitric oxide synthase (NOS), we further explored the contributions of COX and NOS to β-AR vasodilation in this cohort. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured and forearm vascular conductance (FVC) was calculated (FVC = FBF/MAP). The rise in FVC from baseline (ΔFVC) was quantified during graded brachial artery infusion of isoproterenol (Iso, 1-12 ng/100 g/min) in normal weight ( = 36) and adults with obesity ( = 22) (18-40 yr old). In a subset of participants, Iso-mediated vasodilation was examined before and during inhibition of NOS [-monomethyl-l-arginine (l-NMMA)], COX (ketorolac), and NOS + COX (l-NMMA + ketorolac). Iso-mediated increases in FVC did not differ between groups ( = 0.57). l-NMMA attenuated Iso-mediated ΔFVC in normal weight ( = 0.03) but not adults with obesity ( = 0.27). In normal weight adults, ketorolac increased Iso-mediated ΔFVC ( < 0.01) and this response was lost with concurrent l-NMMA ( = 0.67). In contrast, neither ketorolac ( = 0.81) nor ketorolac + l-NMMA ( = 0.40) altered Iso-mediated ΔFVC in adults with obesity. Despite shifts in COX and NOS, β-AR vasodilation is preserved in young adults with obesity. These data highlight the presence of a compensatory shift in microvascular control mechanisms in younger humans with obesity. We examined β-adrenergic receptor-mediated vasodilation in skeletal muscle of humans with obesity and normal weight. Results show that despite shifts in the contribution of cyclooxygenase and nitric oxide synthase, β-adrenergic-mediated vasodilation is relatively preserved in young, otherwise healthy adults with obesity. These data highlight the presence of subclinical changes in microvascular control mechanisms early in the obesity process and suggest duration of obesity and/or the addition of primary aging may be necessary for overt dysfunction.
Topics: Adrenergic beta-Agonists; Adult; Blood Vessels; Cyclooxygenase Inhibitors; Female; Humans; Isoproterenol; Ketorolac; Male; Muscle, Skeletal; Nitric Oxide Synthase Type III; Obesity; Prostaglandin-Endoperoxide Synthases; Receptors, Adrenergic, beta; Vasodilation; omega-N-Methylarginine
PubMed: 34738833
DOI: 10.1152/ajpheart.00449.2021 -
Journal of the American Society For... Jul 2019Arginine methylation is a common protein post-translational modification (PTM) that plays a key role in eukaryotic cells. Three distinct types of this modification are...
Arginine methylation is a common protein post-translational modification (PTM) that plays a key role in eukaryotic cells. Three distinct types of this modification are found in mammals: asymmetric NN-dimethylarginine (aDMA), symmetric NN-dimethylarginine (sDMA), and an intermediate N-monomethylarginine (MMA). Elucidation of regulatory mechanisms of arginine methylation in living organisms requires precise information on both the type of the modified residues and their location inside the protein amino acid sequences. Despite mass spectrometry (MS) being the method of choice for analysis of multiple protein PTMs, unambiguous characterization of protein arginine methylation may not be always straightforward. Indeed, frequent internal basic residues of Arg methylated tryptic peptides hamper their sequencing under positive ion mode collision-induced dissociation (CID), the standardly used tandem mass spectrometry method, while the relative stability of the aDMA and sDMA side chains under alternative non-ergodic electron-based fragmentation techniques, electron-capture and electron transfer dissociations (ECD and ETD), may impede differentiation between the isobaric residues. Here, for the first time, we demonstrate the potential of the negative ion mode collision-induced dissociation MS for analysis of protein arginine methylation and present data revealing that the negative polarity approach can deliver both an unambiguous identification of the arginine methylation type and extensive information on the modified peptide sequences.
Topics: Amino Acid Sequence; Arginine; Peptides; Protein Processing, Post-Translational; Proteins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; omega-N-Methylarginine
PubMed: 30915654
DOI: 10.1007/s13361-019-02176-9 -
PloS One 2017Nitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with...
RATIONALE
Nitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known.
OBJECTIVE
Our objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity.
METHODS AND MEASUREMENTS
34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24-72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay.
MAIN RESULTS
Of all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis.
CONCLUSIONS
In early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo.
Topics: Adult; Aged; Amines; Animals; Arginine; Cell Line; Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Mass Spectrometry; Metabolomics; Mice; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Sepsis; omega-N-Methylarginine
PubMed: 28813479
DOI: 10.1371/journal.pone.0183025 -
Tumour Virus Research Jun 2023Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus...
Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Here, we show that KS lesions express iNOS/NOS2 broadly throughout KS lesions, with enrichment in LANA positive spindle cells. The iNOS byproduct 3-nitrotyrosine is also enriched in LANA positive tumor cells and colocalizes with a fraction of LANA-nuclear bodies. We show that iNOS is highly expressed in the L1T3/mSLK tumor model of KS. iNOS expression correlated with KSHV lytic cycle gene expression, which was elevated in late-stage tumors (>4 weeks) but to a lesser degree in early stage (1 week) xenografts. Further, we show that L1T3/mSLK tumor growth is sensitive to an inhibitor of nitric oxide, L-NMMA. L-NMMA treatment reduced KSHV gene expression and perturbed cellular gene pathways relating to oxidative phosphorylation and mitochondrial dysfunction. These finding suggest that iNOS is expressed in KSHV infected endothelial-transformed tumor cells in KS, that iNOS expression depends on tumor microenvironment stress conditions, and that iNOS enzymatic activity contributes to KS tumor growth.
Topics: Animals; Humans; Mice; Antigens, Viral; Herpesvirus 8, Human; omega-N-Methylarginine; Sarcoma, Kaposi; Tumor Microenvironment
PubMed: 36863485
DOI: 10.1016/j.tvr.2023.200259 -
The Journal of Physiology Mar 2022Cerebrovascular CO reactivity (CVR) is often considered a bioassay of cerebrovascular endothelial function. We recently introduced a test of cerebral shear-mediated...
Cerebrovascular CO reactivity (CVR) is often considered a bioassay of cerebrovascular endothelial function. We recently introduced a test of cerebral shear-mediated dilatation (cSMD) that may better reflect endothelial function. We aimed to determine the nitric oxide (NO)-dependency of CVR and cSMD. Eleven volunteers underwent a steady-state CVR test and transient CO test of cSMD during intravenous infusion of the NO synthase inhibitor N -monomethyl-l-arginine (l-NMMA) or volume-matched saline (placebo; single-blinded and counter-balanced). We measured cerebral blood flow (CBF; duplex ultrasound), intra-arterial blood pressure and . Paired arterial and jugular venous blood sampling allowed for the determination of trans-cerebral NO exchange (ozone-based chemiluminescence). l-NMMA reduced arterial NO by ∼25% versus saline (74.3 ± 39.9 vs. 98.1 ± 34.2 nM; P = 0.03). The steady-state CVR (20.1 ± 11.6 nM/min at baseline vs. 3.2 ± 16.7 nM/min at +9 mmHg ; P = 0.017) and transient cSMD tests (3.4 ± 5.9 nM/min at baseline vs. -1.8 ± 8.2 nM/min at 120 s post-CO ; P = 0.044) shifted trans-cerebral NO exchange towards a greater net release (a negative value indicates release). Although this trans-cerebral NO release was abolished by l-NMMA, CVR did not differ between the saline and l-NMMA trials (57.2 ± 14.6 vs. 54.1 ± 12.1 ml/min/mmHg; P = 0.49), nor did l-NMMA impact peak internal carotid artery dilatation during the steady-state CVR test (6.2 ± 4.5 vs. 6.2 ± 5.0% dilatation; P = 0.960). However, l-NMMA reduced cSMD by ∼37% compared to saline (2.91 ± 1.38 vs. 4.65 ± 2.50%; P = 0.009). Our findings indicate that NO is not an obligatory regulator of steady-state CVR. Further, our novel transient CO test of cSMD is largely NO-dependent and provides an in vivo bioassay of NO-mediated cerebrovascular function in humans. KEY POINTS: Emerging evidence indicates that a transient CO stimulus elicits shear-mediated dilatation of the internal carotid artery, termed cerebral shear-mediated dilatation. Whether or not cerebrovascular reactivity to a steady-state CO stimulus is NO-dependent remains unclear in humans. During both a steady-state cerebrovascular reactivity test and a transient CO test of cerebral shear-mediated dilatation, trans-cerebral nitrite exchange shifted towards a net release indicating cerebrovascular NO production; this response was not evident following intravenous infusion of the non-selective NO synthase inhibitor N -monomethyl-l-arginine. NO synthase blockade did not alter cerebrovascular reactivity in the steady-state CO test; however, cerebral shear-mediated dilatation following a transient CO stimulus was reduced by ∼37% following intravenous infusion of N -monomethyl-l-arginine. NO is not obligatory for cerebrovascular reactivity to CO , but is a key contributor to cerebral shear-mediated dilatation.
Topics: Carbon Dioxide; Cerebrovascular Circulation; Dilatation; Enzyme Inhibitors; Humans; Nitric Oxide; Nitric Oxide Synthase; Nitrogen Dioxide; omega-N-Methylarginine
PubMed: 34904229
DOI: 10.1113/JP282427 -
Diabetes & Vascular Disease Research May 2017RhoA/Rho-associated kinase and arginase are implicated in vascular complications in diabetes. This study investigated whether RhoA/Rho-associated kinase and arginase... (Comparative Study)
Comparative Study
AIM
RhoA/Rho-associated kinase and arginase are implicated in vascular complications in diabetes. This study investigated whether RhoA/Rho-associated kinase and arginase inhibition protect from myocardial ischaemia-reperfusion injury in type 1 diabetes and the mechanisms behind these effects.
METHODS
Rats with streptozotocin-induced type 1 diabetes and non-diabetic rats were subjected to 30 min myocardial ischaemia and 2 h reperfusion after being randomized to treatment with (1) saline, (2) RhoA/Rho-associated kinase inhibitor hydroxyfasudil, (3) nitric oxide synthase inhibitor N-monomethyl-l-arginine monoacetate followed by hydroxyfasudil, (4) arginase inhibitor N-omega-hydroxy-nor-l-arginine, (5) N-monomethyl-l-arginine monoacetate followed by N-omega-hydroxy-nor-l-arginine or (6) N-monomethyl-l-arginine monoacetate given intravenous before ischaemia.
RESULTS
Myocardial arginase activity, arginase 2 expression and RhoA/Rho-associated kinase activity were increased in type 1 diabetes ( p < 0.05). RhoA/Rho-associated kinase inhibition and arginase inhibition significantly reduced infarct size in diabetic and non-diabetic rats ( p < 0.001). The cardioprotective effects of hydroxyfasudil and N-omega-hydroxy-nor-l-arginine in diabetes were abolished by nitric oxide synthase inhibition. RhoA/Rho-associated kinase inhibition attenuated myocardial arginase activity in diabetic rats via a nitric oxide synthase-dependent mechanism.
CONCLUSION
Inhibition of either RhoA/Rho-associated kinase or arginase protects from ischaemia-reperfusion injury in rats with type 1 diabetes via a nitric oxide synthase-dependent pathway. These results suggest that inhibition of RhoA/Rho-associated kinase and arginase constitutes a potential therapeutic strategy to protect the diabetic heart against ischaemia-reperfusion injury.
Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Arginase; Arginine; Cytoprotection; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Drug Therapy, Combination; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide Synthase; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Signal Transduction; omega-N-Methylarginine; rho GTP-Binding Proteins; rho-Associated Kinases
PubMed: 28183205
DOI: 10.1177/1479164116687935