ABSTRACT
Loss of function mutations in store-operated Ca2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1) hypomorph mouse model was generated with significant reduction in Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.
Subject(s)
Hypertension , Hypohidrosis , Animals , Child , Humans , Mice , Calcium/metabolism , Calcium Signaling , Cytokines/metabolism , ORAI1 Protein/genetics , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/metabolism , Cardiovascular System/metabolismABSTRACT
Prostacyclin (PGI2) mimetics (iloprost, treprostinil) are potent vasodilators (primarily via IP-receptor activation) and major therapeutic interventions for pulmonary hypertension (PH). Increased plasma levels of endothelin (ET-1), thromboxane (TxA2) and catecholamines have been demonstrated from patients with PH. In this study, we aimed to compare relaxant effects of iloprost and treprostinil on human (HPA) and rat pulmonary arteries precontracted with either ET-1, thromboxane (U46619) or an α-adrenergic receptor agonist (Norepinephrine, NE or phenylephrine, PE). Treprostinil and iloprost induced vasorelaxation of HPA precontracted with NE, ET-1 or U46619. We obtained greater relaxation response and sensitivity to treprostinil when ET-1 or U46619 were used to induce the precontraction in comparison to NE. In contrast, iloprost showed less relaxation response and sensitivity in HPA precontracted with U46619 versus NE. In the rat, treprostinil and iloprost induced vasorelaxation of pulmonary arteries precontracted with PE and U46619 but minimally with ET-1. However, in rat pulmonary arteries, PE-induced precontractions were comparatively low amplitude. Our study showed that the ex vivo relaxation or sensitivity of pulmonary arteries induced by PGI2 mimetics is highly dependent on both the pre-contraction agent and the species. To best extrapolate to effects on human tissue, our results suggest that U46619 is the appropriate contractile agent for assessing the relaxant effect of PGI2 mimetics in rat pulmonary arteries. Finally we suggest that in PH patients with high plasma concentration of TxA2, treprostinil (not iloprost) would be a preferential treatment. On the other hand, if the ET-1 plasmatic level is high, either treprostinil or iloprost will be effective.
Subject(s)
Epoprostenol/analogs & derivatives , Iloprost/pharmacology , Pulmonary Artery/drug effects , Pulmonary Artery/physiology , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Adrenergic alpha-Agonists/pharmacology , Animals , Epoprostenol/pharmacology , Female , Humans , Male , Rats , Rats, Sprague-DawleyABSTRACT
The current study examined the responsiveness of blood vessels from diabetic rats to K+ channel openers and explored whether ROS might be involved in any changes. Responses were measured in aortic rings isolated from four weeks streptozotocin (65 mg/kg)-induced diabetic rats. Relaxation to levcromakalim (ATP-sensitive potassium channel KATP opener, 10(-9)-10(-5) mol/l) and (+/-)-naringenin (large conductance calcium-activated channel BKCa opener, 10(-8)-10(-3) mol/l) were recorded in phenylephrine (1 µmol/l) pre-contracted segments in the absence and presence of superoxide dismutase (SOD, 100 µmol/l) and apocynin (an antioxidant and inhibitor of NADPH oxidase, 100 µmol/l). Contractions to phenylephrine (10(-9)-10(-5) mol/l) and relaxation to acetylcholine (ACh, 10(-9)-10(-5) mol/l) were also recorded. Relaxation curves for levcromakalim, naringenin and ACh for the diabetic group were shifted to the right (p < 0.05) compared with the control. Contractions to phenylephrine were enhanced in the diabetic group (p < 0.01). SOD restored the ACh response but not those of K+ channel openers. On the other hand, apocynin restored the relaxation to naringenin but had no effect on both levcromakalim and ACh responses. The results suggest that both KATP and BKCa activities are attenuated in diabetes mellitus and that ROS appears to contribute only to the change in BKCa function.
Subject(s)
Blood Vessels/drug effects , Blood Vessels/physiopathology , Diabetes Mellitus/physiopathology , Ion Channel Gating/drug effects , Potassium Channels/metabolism , Reactive Oxygen Species/metabolism , Acetophenones/pharmacology , Acetylcholine/pharmacology , Animals , Blood Glucose/metabolism , Body Weight/drug effects , Cromakalim/pharmacology , Diabetes Mellitus/metabolism , Flavanones/pharmacology , Free Radical Scavengers/pharmacology , Male , Phenylephrine/pharmacology , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/pharmacology , Thiobarbituric Acid Reactive Substances/metabolism , Vasoconstriction/drug effects , Vasodilation/drug effectsABSTRACT
OBJECTIVE: Excessive opening of the adenosine triphosphate-sensitive potassium channel in vascular smooth muscle is implicated in the vasodilation and vascular hyporeactivity underlying septic shock. Therapeutic channel inhibition using sulfonylurea agents has proved disappointing, although agents acting on its pore appear more promising. We thus investigated the hemodynamic effects of adenosine triphosphate-sensitive potassium channel pore inhibition in awake, fluid-resuscitated septic rats, and the extent to which these responses are modulated by the high sympathetic tone present in sepsis. Temporal changes in ex-vivo channel activity and subunit gene expression were also investigated. DESIGN: In vivo and ex vivo animal study. SETTING: University research laboratory. SUBJECTS: Male adult Wistar rats. INTERVENTIONS AND MEASUREMENTS: Fecal peritonitis was induced in conscious, fluid-resuscitated rats. Pressor responses to norepinephrine and PNU-37883A (a vascular adenosine triphosphate-sensitive potassium channel inhibitor acting on the Kir6.1 pore-forming subunit) were measured at 6 or 24 hrs, in the absence or presence of the autonomic ganglion blocker, pentolinium. The aorta and mesenteric artery were examined ex vivo for rubidium efflux as a marker of adenosine triphosphate-sensitive potassium channel activity, and for adenosine triphosphate-sensitive potassium channel subunit gene expression using quantitative reverse transcription-polymerase chain reaction. MAIN RESULTS: A total of 120 rats (50 sham-operated controls, 70 septic) were included. Septic rats became hypotensive after 12 hrs, with a 24-hr mortality of 51.7% (0% in controls). At 6 hrs, there was an attenuated pressor response to norepinephrine (p < .01) despite blood pressure being elevated (p < .01). PNU-37883A had no pressor effect, except in the presence of pentolinium (p < .01). Kir6.1 subunit mRNA increased significantly in the mesenteric artery while rubidium efflux was increased in both the aorta and mesenteric artery at 24 hrs. CONCLUSIONS: Despite evidence of increased adenosine triphosphate-sensitive potassium channel activity in sepsis, it appears to be inhibited in vivo by high sympathetic tone. This may explain, at least in part, the reduced efficacy of adenosine triphosphate-sensitive potassium channel blockers in human septic shock.
Subject(s)
Blood Vessels/physiopathology , Sepsis/physiopathology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Adamantane/analogs & derivatives , Adamantane/pharmacology , Animals , Aorta/drug effects , Aorta/physiopathology , Blood Vessels/drug effects , Blood Vessels/metabolism , Ganglionic Blockers/pharmacology , Gene Expression/drug effects , Gene Expression/physiology , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiopathology , Morpholines/pharmacology , Norepinephrine/pharmacology , Pentolinium Tartrate/pharmacology , Rats , Rats, Wistar , Sepsis/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium-Potassium-Exchanging ATPase/physiology , Vasodilation/drug effects , Vasodilation/physiologyABSTRACT
AIMS: Norepinephrine (NE) is a known regulator of adipose tissue (AT) metabolism, angiogenesis, vasoconstriction and fibrosis. This may be through autocrine/paracrine effects on local resistance vessel function and morphology. The aims of this study were to investigate, in human subcutaneous and omental adipose tissue (SAT and OAT): NE synthesis, angiogenesis, NE-mediated arteriolar vasoconstriction, the induction of collagen gene expression and its deposition in non-diabetic versus diabetic obese subjects. MATERIALS AND METHODS: SAT and OAT from obese patients were used to investigate tissue NE content, tyrosine hydroxylase (TH) density, angiogenesis including capillary density, angiogenic capacity and angiogenic gene expression, NE-mediated arteriolar vasoconstriction and collagen deposition. KEY FINDINGS: In the non-diabetic group, NE concentration, TH immunoreactivity, angiogenesis and maximal vasoconstriction were significantly higher in OAT compared to SAT (p < 0.05). However, arterioles from OAT showed lower NE sensitivity compared to SAT (10-8 M to 10-7.5 M, p < 0.05). A depot-specific difference in collagen deposition was also observed, being greater in OAT than SAT. In the diabetic group, no significant depot-specific differences were seen in NE synthesis, angiogenesis, vasoconstriction or collagen deposition. SAT arterioles showed significantly lower sensitivity to NE (10-8 M to 10-7.5 M, p < 0.05) compared to the non-diabetic group. SIGNIFICANCE: SAT depot in non-diabetic obese patients exhibited relatively low NE synthesis, angiogenesis, tissue fibrosis and high vasoreactivity, due to preserved NE sensitivity. The local NE synthesis in OAT and diabetes desensitizes NE-induced vasoconstriction, and may also explain the greater tissue angiogenesis and fibrosis in these depots.
Subject(s)
Diabetes Mellitus , Neovascularization, Pathologic , Norepinephrine , Adipose Tissue/metabolism , Collagen/metabolism , Diabetes Mellitus/metabolism , Fibrosis , Humans , Neovascularization, Pathologic/metabolism , Norepinephrine/metabolism , Obesity/metabolismABSTRACT
Diabetes in humans a chronic metabolic disorder characterised by hyperglycaemia, it is associated with an increased risk of cardiovascular disease, disruptions to metabolism and vascular functions. It is also linked to oxidative stress and its complications. Its role in vascular dysfunctions is generally reported without detailed impact on the molecular mechanisms. Potassium ion channel (K+ channels) are key regulators of vascular tone, and as membrane proteins, are modifiable by oxidant stress associated with diabetes. This review manuscript examined the impact of oxidant stress on vascular K+ channel functions in diabetes, its implication in vascular complications and metabolic and cardiovascular diseases.
ABSTRACT
Aim: To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. Methods: Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC-MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography. Results: CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition. Conclusion: In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation.
ABSTRACT
Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35−36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p < 0.05 and met ≥ 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT.
ABSTRACT
Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish-peroxidase (HRP)-conjugated anti-EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non-specific cross-reactivity with secondary antibodies. An in-house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti-human EPO-HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in-house anti-EPO-HRP conjugate was performed as well as the current two-step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in-house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti-EPO-HRP conjugate showed comparable specificity but had lower sensitivity to both the in-house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP-conjugated anti-EPO antibody to reduce analysis time for EPO in doping control.
Subject(s)
Antibodies/immunology , Erythropoietin/analysis , Horseradish Peroxidase/immunology , Substance Abuse Detection/methods , Blotting, Western , Doping in Sports/prevention & control , Electrophoresis, Polyacrylamide Gel , Erythropoietin/immunology , Horseradish Peroxidase/chemistry , Humans , Recombinant Proteins , Sensitivity and Specificity , Time FactorsABSTRACT
AIMS: Obesity is a risk factor for endothelial dysfunction, the severity of which is likely to vary depending on extent and impact of adiposity on the vasculature. This study investigates the roles of cyclooxygenase isoforms and thromboxane receptor activities in the differential endothelial dilatory capacities of arteries derived from omental and subcutaneous adipose tissues in obesity. MAIN METHODS: Small arteries were isolated from omental and subcutaneous adipose tissues obtained from consented morbidly obese patients (nâ¯=â¯65, BMI 45⯱â¯6â¯kgâ¯m-2 [Mean⯱â¯SD]) undergoing bariatric surgery. Relaxation to acetylcholine was studied by wire myography in the absence or presence of indomethacin (10⯵M, cyclooxygenase inhibitor), FR122047 (1⯵M, cyclooxygenase-1 inhibitor), Celecoxib (4⯵M, cyclooxygenase-2 inhibitor), Nω-Nitro-L-arginine methyl ester (L-NAME, 100⯵M, nitric oxide synthase inhibitor) or combination of apamin (0.5⯵M) and charybdotoxin (0.1⯵M) that together inhibit endothelium-derived hyperpolarizing factor (EDHF). Contractions to U46619 (thromboxane A2 mimetic) were also studied. KEY FINDINGS: Acetylcholine relaxation was significantly attenuated in omental compared with subcutaneous arteries from same patients (p < 0.01). Indomethacin (p < 0.01) and FR122047 (p < 0.001) but not Celecoxib significantly improved the omental arteriolar relaxation. Cyclooxygenase-1 mRNA and U46619 contractions were both increased in omental compared with subcutaneous arteries (p < 0.05). L-NAME comparably inhibited acetylcholine relaxation in both arteries, while apamin+charybdotoxin were less effective in omental compared with subcutaneous arteries. SIGNIFICANCE: The results show that the depot-specific reduction in endothelial dilatory capacity of omental compared with subcutaneous arteries in obesity is in large part due to altered cyclooxygenase-1 and enhanced thromboxane receptor activities, which cause EDHF deficiency.
Subject(s)
Cyclooxygenase 1/metabolism , Gastroepiploic Artery/drug effects , Receptors, Thromboxane/metabolism , Adipose Tissue/blood supply , Adipose Tissue/metabolism , Adult , Apamin/pharmacology , Arteries/drug effects , Celecoxib/pharmacology , Charybdotoxin/pharmacology , Cyclooxygenase 1/physiology , Cyclooxygenase Inhibitors/pharmacology , Endothelial Cells/metabolism , Endothelial Cells/physiology , Endothelium, Vascular/drug effects , Female , Gastroepiploic Artery/metabolism , Humans , Indomethacin/pharmacology , Male , Middle Aged , Muscle Relaxation/drug effects , NG-Nitroarginine Methyl Ester/pharmacology , Obesity, Morbid/metabolism , Omentum/blood supply , Omentum/metabolism , Receptors, Thromboxane/physiology , Vasodilation/drug effectsABSTRACT
Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release and causing symptoms similar to sepsis, e.g. hypotension and subsequent hyporeactivity. We investigated the direct effect of SAgs on vascular tone using two recombinant SAgs, SEA and SPEA. The roles of Nitric Oxide (NO) and potentially hyperpolarization, which is dependent on the K+ channel activation, were also explored. The data show that SEA and SPEA have direct vasodilatory effects that were in part NO-dependent, but completely dependent on activation of K+ channels. Our work also identified the functional regions of one of the superantigens, SPEA, that are involved in causing the vasodilation and possible hypotension. A series of 20 overlapping peptides, spanning the entire sequence of SPEA, were designed and synthesized. The vascular response of each peptide was measured, and the active peptides were identified. Our results implicate the regions, (61-100), (101-140) and (181-220) which cause the vasodilation and possible hypotension effects of SPEA. The data also shows that the peptide 181-220 exert the highest vasodilation effect. This work therefore, demonstrates the direct effect of SAgs on vascular tone and identify the active region causing this vasodilation. We propose that these three peptides could be effective novel antihypertensive drugs. We also overexpressed, in E.coli, four superantigens from codon optimized genes.
Subject(s)
Lymphocyte Activation/drug effects , Neoplasms/drug therapy , Peptides/pharmacology , Superantigens/pharmacology , T-Lymphocytes/drug effects , Vasodilation/drug effects , Animals , Escherichia coli/genetics , Immunotherapy , Muscle, Smooth, Vascular/drug effects , Neoplasms/immunology , Peptides/genetics , Sheep , Superantigens/genetics , T-Lymphocytes/immunologyABSTRACT
OBJECTIVE: We investigated the role of the inward rectifier potassium (KIR) channel and the cyclic AMP-dependent pathway in mediating vasorelaxation induced by the prostacyclin analogue cicaprost. METHODS: Small vessel myography was used to assess responses to cicaprost in segments of rat tail artery contracted with phenylephrine. Microelectrode recordings were made from helical strips to assess effects on membrane potential. RESULTS: Cicaprost caused relaxation and hyperpolarisation that were significantly inhibited by Ba2+ (30-100 microM), a known blocker of KIR channels. Raising extracellular K+ from 5 to 15 mM elicited membrane hyperpolarisation and an endothelium-independent relaxation that was blocked by Ba2+ (30-100 microM), suggesting the existence of functional KIR channels on the smooth muscle. In contrast, neither glibenclamide (10 microM), a blocker of ATP-sensitive K+ channels, nor fluoxetine hydrochloride (100 microM), a blocker of G-protein-gated inward rectifier K+ channels, nor pertussis toxin (PTX; 1 microg/ml), which irreversibly inhibits Gi/Go, reduced relaxation to cicaprost. Indeed, PTX significantly potentiated responses. Relaxation to cicaprost was not mediated by NO but was partially endothelium-dependent, consistent with a similar inhibition by a combination of charybdotoxin (0.1 microM) and apamin (0.5 microM), blockers of endothelium-derived hyperpolarising factor (EDHF). However, relaxation was unaffected by adenylyl cyclase (SQ22536, dideoxyadenosine) or protein kinase A (Rp-2-O-monobutyryl-cAMP) inhibitors, consistent also with Ba2+ only weakly inhibiting relaxation to the adenylyl cyclase activator forskolin. CONCLUSION: We conclude that cicaprost relaxes rat tail artery by activating KIR channels with some involvement from EDHF. The mechanism appears to be largely independent of cyclic AMP and Gi/Go, although the latter appears to counteract relaxation through an unknown pathway and/or receptor.
Subject(s)
Cyclic AMP/metabolism , Epoprostenol/analogs & derivatives , Muscle, Smooth, Vascular/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Receptors, Epoprostenol/agonists , Vasodilator Agents/pharmacology , Adenine/analogs & derivatives , Adenine/pharmacology , Adenylyl Cyclase Inhibitors , Animals , Apamin/pharmacology , Barium Compounds/pharmacology , Charybdotoxin/pharmacology , Chlorides/pharmacology , Cromakalim/pharmacology , DDT/analogs & derivatives , DDT/pharmacology , Dose-Response Relationship, Drug , Epoprostenol/pharmacology , Fluoxetine/pharmacology , Glyburide/pharmacology , In Vitro Techniques , Male , Neurotoxins/pharmacology , Phenylephrine/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para -hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5+/-0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3+/-0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([(2)H(6)]PHPA) was injected, and the formation of deuterated NHPA ([(2)H(5)]NHPA) was measured. Of the infused [(2)H(6)]PHPA, 78+/-2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [(2)H(5)]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.
Subject(s)
Nitrates/metabolism , Nitrophenols/metabolism , Phenylacetates/metabolism , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Animals , Deuterium/administration & dosage , Deuterium/metabolism , Gas Chromatography-Mass Spectrometry , Humans , Injections, Intravenous , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/pharmacology , Male , Models, Chemical , Nitrophenols/blood , Nitrophenols/urine , Nitrosation , Phenylacetates/blood , Phenylacetates/urine , Rats , Rats, Sprague-Dawley , Reference Standards , Tyrosine/administration & dosage , Tyrosine/pharmacologySubject(s)
Muscle, Smooth, Vascular/physiopathology , Potassium Channels, Calcium-Activated/physiology , Shock, Hemorrhagic/physiopathology , Animals , Blood Pressure/physiology , Calcium/physiology , Electrophysiology , Gene Expression Regulation , Mice , Mice, Knockout , Muscle, Smooth, Vascular/pathology , Potassium Channels, Calcium-Activated/genetics , Protein Subunits/genetics , Protein Subunits/physiologyABSTRACT
Prostanoid IP receptors coupled to Gs are thought to be the primary target for prostacyclin (PGI(2)) analogues. However, these agents also activate prostanoid EP(1-4) receptor subtypes to varying degrees, which are positively (EP(2/4)) or negatively (EP(3)) coupled to adenylate cyclase through Gs or Gi, respectively. We investigated the role of these receptors in modulating relaxation to PGI(2) analogues cicaprost, iloprost and treprostinil in pre-contracted segments of rat tail artery. Prostanoid IP (RO1138452), EP(4) (GW627368X), EP(3) (L-798106), EP(1-3) (AH6809), and EP(1) (SC-51322) receptor antagonists were used to determine each receptor contribution. The role of G(i/o) was investigated using pertussis toxin (PTX), while dependence on cAMP was determined using adenylate cyclase (2'5'dideoxyadenosine, DDA) and protein kinase A (2'-O-monobutyryladenosine- 3',5'-cyclic monophosphorothioate, Rp- isomer, Rp-2'-O-MB-cAMPS) inhibitors, and by measurement of tissue cAMP. All analogues caused relaxation which was significantly (P<0.01) inhibited by RO1138452; with maximum response to cicaprost, iloprost and treprostinil reduced by 51%, 66% and 37%, respectively. GW627368X had no effect when used alone, but in combination with RO1138452, caused a rightward shift of the curves for cicaprost and iloprost but not treprostinil. PTX treatment potentiated relaxation to all 3 analogues (P<0.01), as did L798106 and AH6809 but not SC-51322. Basal cAMP levels were higher in PTX-treated tissues and DDA- and Rp-2'-O-MB-cAMPs--sensitive responses increased to analogue concentrations <0.1µM. In conclusion, prostanoid EP(3) receptors via G(i/o) negatively modulate prostanoid IP receptor-mediated relaxation to cicaprost, iloprost and treprostinil. However, other pathways contribute to analogue-induced vasorelaxation, the nature of which remains unclear for treprostinil.
Subject(s)
Arteries/drug effects , Epoprostenol/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptors, Prostaglandin E, EP3 Subtype/metabolism , Receptors, Prostaglandin/metabolism , Tail/blood supply , Vasodilation/drug effects , Animals , Arteries/metabolism , Arteries/physiology , Cyclic AMP/metabolism , Epoprostenol/analogs & derivatives , In Vitro Techniques , Male , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Epoprostenol , Vasodilator Agents/chemistry , Vasodilator Agents/pharmacologyABSTRACT
Nitric oxide-mediated activation of large conductance calcium-activated potassium (BK) channels is considered an important underlying mechanism of sepsis-induced hypotension. Indeed, the nonselective K-channel inhibitor, tetraethylammonium chloride (TEA), has been proposed as a potential treatment to raise blood pressure in septic shock by virtue of its ability to inhibit BK channels. As experimental evidence has so far relied on pharmacological inhibition, we examined the effects of channel deletion using BKα subunit knockout (α, Slo) mice in two mouse models of polymicrobial sepsis, namely, intraperitoneal fecal slurry and cecal ligation and puncture. Comparison was made against TEA treatment in wild-type (WT) mice. Following slurry, BKα and WT mice developed similar degrees of hypotension over 10 h with no difference in cardiac output as assessed by echocardiography between groups. Tetraethylammonium chloride raised blood pressure significantly in septic WT mice, but had no effect on survival. However, following cecal ligation and puncture, a significantly reduced survival was seen in both BKα mice and (high-dose) TEA-treated WT mice compared with untreated WT animals. In conclusion, the BK channel does not appear to be integral to sepsis-induced hypotension but does affect survival through other mechanisms. The pressor effect of TEA may be related to effects on other potassium channels.
Subject(s)
Hypotension/genetics , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/deficiency , Sepsis/microbiology , Sepsis/mortality , Animals , Blood Pressure/drug effects , Blood Pressure/genetics , Genotype , Hypotension/drug therapy , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/genetics , Male , Mice , Mice, Knockout , Potassium Channel Blockers/therapeutic use , Reverse Transcriptase Polymerase Chain Reaction , Sepsis/drug therapy , Sepsis/genetics , Tetraethylammonium/therapeutic useABSTRACT
OBJECTIVE: There is escalating interest in the therapeutic use of vasopressin in septic shock. However, little attention has focused on mechanisms underlying its pressor hypersensitivity, which contrasts with the vascular hyporesponsiveness to catecholamines. We investigated whether a long-term rodent model of sepsis would produce changes in endogenous levels and pressor reactivity to exogenous norepinephrine and vasopressin comparable with those seen in septic patients. DESIGN: In vivo and ex vivo animal study. SETTING: University research laboratory. SUBJECTS: Male adult Wistar rats. INTERVENTIONS AND MEASUREMENTS: Fecal peritonitis was induced in conscious, fluid-resuscitated rats. Biochemical and hormonal profiles were measured at time points up to 48 hrs. Pressor responses to intravenous norepinephrine, vasopressin, and F-180, a selective V1 receptor agonist, were measured at 24 hrs. Contractile responses to these drugs were assessed in mesenteric arteries taken from animals at 24 hrs using wire myography. Comparisons were made against sham operation controls. MAIN RESULTS: Septic rats became unwell and hypotensive, with a mortality of 64% at 48 hrs (0% in controls). Plasma norepinephrine levels were elevated in septic animals at 24 hrs (1968 +/- 490 vs. 492 +/- 90 pg/mL in controls, p = .003), whereas vasopressin levels were similar in the two groups (4.5 +/- 0.8 vs. 3.0 +/- 0.5 pg/mL, p = not significant). In vivo, the pressor response to norepinephrine was markedly reduced in the septic animals, but responses to vasopressin and F-180 were relatively preserved. In arteries from septic animals, norepinephrine contractions were decreased (efficacy as measured by maximum contractile response, Emax: 3.0 +/- 0.3 vs. 4.7 +/- 0.2 mN, p < .001). In contrast, the potency of vasopressin (expressed as the negative log of the concentration required to produce 50% of the maximum tension, pD2: 9.1 +/- 0.04 vs. 8.7 +/- 0.05, p < .001) and F-180 (pD2 8.2 +/- 0.04 vs. 7.6 +/- 0.02, p < .001) was enhanced (n > or = 6 for all groups). CONCLUSIONS: This long-term animal model demonstrates changes in circulating vasoactive hormones similar to prolonged human sepsis, and decreased pressor sensitivity to norepinephrine. Ex vivo sensitivity to vasopressin agonists was heightened. This model is therefore appropriate for the further investigation of mechanisms underlying vasopressin hypersensitivity, which may include receptor or calcium-handling alterations within the vasculature.
Subject(s)
Norepinephrine/therapeutic use , Shock, Septic/drug therapy , Vasoconstrictor Agents/therapeutic use , Vasomotor System/drug effects , Vasopressins/therapeutic use , Animals , Disease Models, Animal , Norepinephrine/blood , Rats , Rats, Wistar , Time Factors , Vasopressins/bloodABSTRACT
It is now established that S-nitroso-albumin (SNO-albumin) circulates at low nanomolar concentrations under physiological conditions, but concentrations may increase to micromolar levels during disease states (e.g., cirrhosis or endotoxemia). This study tested the hypothesis that high concentrations of SNO-albumin observed in some diseases modulate vascular function and that it acts as a stable reservoir of nitric oxide (NO), releasing this molecule when the concentrations of low-molecular-weight thiols are increased. SNO-albumin was infused into rats to increase the plasma concentration from <50 nmol/l to approximately 4 micromol/l. This caused a 29 +/- 6% drop in blood pressure, 20 +/- 4% decrease in aortic blood flow, and a 25 +/- 14% reduction of renal blood flow within 10 min. These observations were in striking contrast to those of an infused arterial vasodilator (hydralazine), which increased aortic blood flow, and suggested that SNO-albumin acts primarily as a venodilator in vivo. This was confirmed by the observations that glyceryl trinitrate (a venodilator) led to similar hemodynamic changes and that the hemodynamic effects of SNO-albumin are reversed by infusion of colloid. Infusion of N-acetylcysteine into animals with artificially elevated plasma SNO-albumin concentrations led to the rapid decomposition of SNO-albumin in vivo and reproduced the hemodynamic effects of SNO-albumin infusion. These data demonstrate that SNO-albumin acts primarily as a venodilator in vivo and represents a stable reservoir of NO that can release NO when the concentrations of low-molecular-weight thiols are elevated.