Search Results (34)

Recent bioassay studies have unexpectedly supported the high (computationally predicted) binding affinities of angiotensin receptor blockers (ARBs) at α-adrenergic receptors (αARs) in isolated smooth muscle. Computational predictions from ligand docking studies are consistent with very low concentrations of ARBs (e.g., sartans or bisartans) that partially reduce (20–50%) the contractile response to phenylephrine, suggesting that some ARBs may function as partial inverse agonists at αARs. Virtual ligand screening (docking) and molecular dynamics (MD) simulations were carried out to explore the binding affinities and stabilities of selected non-peptide ligands (e.g., ARBs and small-molecule opioids) for several G-protein coupled receptor (GPCR) types, including angiotensin II (AngII) type 1 receptor (AT₁R), α1AR, α2AR, and μ-(µOR) and ժ-opioid receptors (ժOR). Results: All ligands docked preferentially to the binding pocket on the cell surface domain of the GPCR types investigated. Drug binding was characterized by weak interactions (hydrophobic, hydrogen bonding, pi-pi) and stronger ionic and salt-bridge interactions (cation-pi and cation-anion interactions). Ligands specific to each GPCR category showed considerable cross-binding with alternative GPCRs, with small-molecule medications appearing less selective than their peptide or ARB functional equivalents. ARBs that exhibit higher affinities for AT₁R also demonstrate higher affinities for µORs and ժORs than opiate ligands, such as fentanyl and naltrexone. Moreover, ARBs had a higher affinity for αARs than either alpha agonists (epinephrine and phenylephrine) or inhibitors (prazosin and doxazosin). MD simulations of membrane-embedded ARB-GPCR complexes proved stable over nanosecond time scales and suggested that some ARBs may behave as agonists or antagonists depending on the GPCR type. Based on the results presented in this and related investigations, we propose that agonists bind to the resting A-site of GPCRs, while inverse agonists occupy the desensitizing D-site, which partial agonists like morphine and fentanyl share, contributing to addiction. ARBs block both AngII and alpha receptors, suggesting that they are more potent antihypertensive drugs than ACE inhibitors. ARBs have the potential to inhibit morphine tolerance and appear to disrupt receptor desensitization processes, potentially by competing at the D-site. Our results suggest the possible therapeutic potential of ARBs in treating methamphetamine and opiate addictions. Full article

For the interaction of angiotensin II (AngII) with AngII type 1 receptors (AT₁R), two potential proton hopping pathways have been identified, each associated with distinct physiological outcomes. The octapeptide AngII (Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸) appears to form a charge relay system (CRS) in solution in which the C-terminal carboxylate abstracts a proton from the His⁶ imidazole group, which, in turn, abstracts a proton from the Tyr⁴ hydroxyl (OH) group, creating a tyrosinate anion. When AngII binds to the AT₁R, the CRS can be reconstituted with D281 of the receptor taking up the role of the Phe⁸ carboxylate in the tripartite interaction, whilst the Phe⁸ carboxylate forms a salt bridge with K199 of the receptor. As a consequence, the Tyr⁴ OH of AngII is positioned with accessibility to either the Phe⁸ carboxylate (bound to K199) or the His⁶ imidazole (activated by D281), thereby creating a potential gating mechanism for AT₁R receptor signaling. This study summarizes evidence based on structure activity data for various analogs wherein Tyr⁴ OH interaction with His⁶ imidazole (CRS formation) leads to G protein sequestration and vasoconstriction, whereas Tyr⁴ OH interaction with Phe⁸ carboxylate (bound to K199) engenders arrestin-mediated vasodilation and receptor desensitization. These findings, combined with quantum mechanical (semiempirical) calculations of CRS proton transfer presented herein, provide insights for the therapeutic targeting of angiotensin receptor blockers (sartans) and the development of second-generation drugs (bisartans). Full article

The octapeptide angiotensin II (Ang II) is a circulating hormone as well as a locally formed agonist synthesized by the angiotensin-converting enzyme (ACE) of endothelial cells. It forms a powerful mechanism to control the amount and pressure of body fluids. All main effects are directed to save body salt and water and ensure blood pressure under basic conditions and in emergencies. All blood vessels respond to stimulation by Ang II; the immediate response is smooth muscle contraction, increasing vascular resistance, and elevating blood pressure. Such effects are conveyed by type 1 angiotensin receptors (AT₁Rs) located in the plasma membrane of both endothelial and vascular smooth muscle cells. AT₁Rs are heterotrimeric G protein-coupled receptors (GPCRs), but their signal pathways are much more complicated than other GPCRs. In addition to G_q/11, the G_12/13, JAK/STAT, Jnk, MAPK, and ERK 1/2, and arrestin-dependent and -independent pathways are activated because of the promiscuous attachment of different signal proteins to the intracellular G protein binding site and to the intracellular C terminal loop. Substantial changes in protein expression follow, including the intracellular inflammation signal protein NF-κB, endothelial contact proteins, cytokines, matrix metalloproteinases (MMPs), and type I protocollagen, eliciting the inflammatory transformation of endothelial and vascular smooth muscle cells and fibrosis. Ang II is an important contributor to vascular pathologies in hypertensive, atherosclerotic, and aneurysmal vascular wall remodeling. Such direct vascular effects are reviewed. In addition to reducing blood pressure, AT₁R antagonists and ACE inhibitors have a beneficial effect on the vascular wall by inhibiting pathological wall remodeling. Full article

Background/Objectives: Increased sodium chloride (NaCl) intake led to leukocyte activation and impaired vasodilatation via increased oxidative stress in human/animal models. Interestingly, subpressor doses of angiotensin II (AngII) restored endothelium-dependent vascular reactivity, which was impaired in a high-salt (HS) diet in animal models. Therefore, the present study aimed to assess the effects of AngII exposure following high salt (HS) loading on endothelial cells’ (ECs’) viability, activation, and reactive oxygen species (ROS) production. Methods: The fifth passage of human aortic endothelial cells (HAECs) was cultured for 24, 48, and 72 h with NaCl, namely, the control (270 mOsmol/kg), HS320 (320 mOsmol/kg), and HS350 (350 mOsmol/kg). AngII was administered at the half-time of the NaCl incubation (10⁻⁴–10⁻⁷ mol/L). Results: The cell viability was significantly reduced after 24 h in the HS350 group and in all groups after longer incubation. AngII partly preserved the viability in the HAECs with shorter exposure and lower concentrations of NaCl. Intracellular hydrogen peroxide (H₂O₂) and peroxynitrite (ONOO⁻) significantly increased in the HS320 group following AngII exposure compared to the control, while it decreased in the HS350 group compared to the HS control. A significant decrease in superoxide anion (O₂^.−) formation was observed following AngII exposure at 10⁻⁵, 10⁻⁶, and 10⁻⁷ mol/L for both HS groups. There was a significant decrease in intracellular adhesion molecule 1 (ICAM-1) and endoglin expression in both groups following treatment with 10⁻⁴ and 10⁻⁵ mol/L of AngII. Conclusions: The results demonstrated that AngII significantly reduced ROS production at HS350 concentrations and modulated the viability, proliferation, and activation states in ECs. Full article

Alterations in cellular energy metabolism are a hallmark of cancer and lactate dehydrogenase (LDH) enzymes are overexpressed in many cancers regardless of sufficient oxygen and functional mitochondria. Further, L-lactate plays signaling roles in multiple cell types. We evaluated the effect of singlet oxygen and hypochlorous acid (HOCl) on pig heart LDH-B, which shares 97% homology with human LDH-B. Singlet oxygen was generated photochemically using methylene blue or the chlorophyll metabolites, pheophorbide A and chlorin e6. Singlet oxygen induced protein crosslinks observed by SDS-PAGE under reducing conditions and inhibited LDH-B activity. Ascorbate, hydrocaffeic acid, glutathione and sodium azide were employed as singlet oxygen scavengers and shown to protect LDH-B. Using fluorescein-modified maleimide, no changes in cysteine availability as a result of singlet oxygen damage were observed. This was in contrast to HOCl, which induced the formation of disulfides between LDH-B subunits, thereby decreasing LDH-B labeling with fluorescein. HOCl oxidation inhibited LDH-B activity; however, disulfide reduction did not restore it. LDH-B cysteines were resistant to millimolar H₂O₂, chloramines and Angeli’s salt. In the absence of pyruvate, LDH-B enhanced NADH oxidation in a chain reaction initiated by singlet oxygen that resulted in H₂O₂ formation. Once damaged by either singlet oxygen or HOCl, NADH oxidation by LDH-B was impaired. Full article

Muscle lactate dehydrogenase (LDH-A) catalyzes the reduction of pyruvate to lactate, the end product of anaerobic glycolysis. LDH-A is overexpressed in many cancers prior to and even when tumors receive adequate oxygen, and lactate has multiple cellular roles. We assessed the effect of singlet oxygen and hypochlorous acid (HOCl) on mammalian LDH-A. Oxidants induced distinct patterns of protein crosslinks observed by SDS-PAGE under reducing conditions. LDH-A cysteines were detected using fluorescein-modified maleimide to assess their oxidation and accessibility. Singlet oxygen initially increased cysteine exposure, but higher doses resulted in their oxidation in addition to non-reducible covalent crosslinks. LDH-A cysteines were oxidized by micromolar HOCl (1–10 equivalents over enzyme) but were resistant to millimolar H₂O₂, chloramines and Angeli’s salt. HOCl oxidation inhibited LDH-A activity and yielded inter-chain disulfides observed by nonreducing SDS-PAGE. Disulfide reduction did not restore LDH-A activity that was lost due to HOCl oxidation. An irreversible conformational change induced by HOCl was detected by native gel electrophoresis and tryptophan fluorescence. In the absence of pyruvate, LDH-A enhanced NADH oxidation resulting in H₂O₂ formation. Singlet oxygen, but not HOCl, initiated this superoxide-dependent chain reaction. Once damaged by both singlet oxygen or HOCl, LDH-A had decreased NADH oxidation activity. Full article

Dinitrosyl iron complexes (DNICs) are important physiological derivatives of nitric oxide. These complexes have a wide range of biological activities, with antioxidant and antiradical ones being of particular interest and importance. We studied the interaction between DNICs associated with the dipeptide L-carnosine or serum albumin and prooxidants under conditions mimicking oxidative stress. The ligands of these DNICs were histidine residues of carnosine or His39 and Cys34 in bovine serum albumin. Carnosine-bound DNICs reduced the level of piperazine free radicals in the reaction system containing tert-butyl hydroperoxide (t-BOOH), bivalent iron ions, a nitroxyl anion donor (Angeli’s salt), and HEPES buffer. The ability of carnosine DNICs to intercept organic free radicals produced from t-BOOH decay could lead to this effect. In addition, carnosine DNICs reacted with the superoxide anion radical (O₂^•−) formed in the xanthine/xanthine oxidase enzymatic system. They also reduced the oxoferryl form of the heme group formed in the reaction of myoglobin with t-BOOH. DNICs associated with serum albumin were found to be rapidly destroyed in a model system containing metmyoglobin and t-BOOH. At the same time, these protein DNICs inhibited the t-BOOH-induced oxidative degradation of coenzymes Q₉ and Q₁₀ in rat myocardial homogenate. The possible mechanisms of the antioxidant and antiradical action of the DNICs studied and their role in the metabolism of reactive oxygen and nitrogen species are discussed. Full article

Chagas disease (CD), caused by Trypanosoma cruzi, is a neglected tropical disease prevalent in Latin America. Infected patients are treated to eliminate the parasite, reduce the cardiomyopathy risk, and interrupt the disease transmission cycle. The World Health Organization recognizes benznidazole (BZ) and nifurtimox as effective drugs for CD treatment. In the chronic phase, both drugs have low cure rates and serious side effects. T. cruzi infection causes intense tissue inflammation that controls parasite proliferation and CD evolution. Compounds that liberate nitric oxide (NO) (NO donors) have been used as anti-T. cruzi therapeutics. Currently, there is no evidence that nitroxyl (HNO) affects T. cruzi infection outcomes. This study investigated the effects of the HNO donor Angeli’s salt (AS) on C57BL/6 mice infected with T. cruzi (Y strain, 5 × 10³ trypomastigotes, intraperitoneally). AS reduced the number of parasites in the bloodstream and heart nests and increased the protective antioxidant capacity of erythrocytes in infected animals, reducing disease severity. Furthermore, in vitro experiments showed that AS treatment reduced parasite uptake and trypomastigote release by macrophages. Taken together, these findings from the murine model and in vitro testing suggest that AS could be a promising therapy for CD. Full article

Congestive heart failure (CHF) is often associated with impaired kidney function. Over- activation of the renin–angiotensin–aldosterone system (RAAS) contributes to avid salt/water retention and cardiac hypertrophy in CHF. While the deleterious effects of angiotensin II (Ang II) in CHF are well established, the biological actions of angiotensin 1-7 (Ang 1-7) are not fully characterized. In this study, we assessed the acute effects of Ang 1-7 (0.3, 3, 30 and 300 ng/kg/min, IV) on urinary flow (UF), urinary Na⁺ excretion (UNaV), glomerular filtration rate (GFR) and renal plasma flow )RPF) in rats with CHF induced by the placement of aortocaval fistula. Additionally, the chronic effects of Ang 1-7 (24 µg/kg/h, via intra-peritoneally implanted osmotic minipumps) on kidney function, cardiac hypertrophy and neurohormonal status were studied. Acute infusion of either Ang 1-7 or its agonist, AVE 0991, into sham controls, but not CHF rats, increased UF, UNaV, GFR, RPF and urinary cGMP. In the chronic protocols, untreated CHF rats displayed lower cumulative UF and UNaV than their sham controls. Chronic administration of Ang 1-7 and AVE 0991 exerted significant diuretic, natriuretic and kaliuretic effects in CHF rats, but not in sham controls. Serum creatinine and aldosterone levels were significantly higher in vehicle-treated CHF rats as compared with controls. Treatment with Ang 1-7 and AVE 0991 reduced these parameters to comparable levels observed in sham controls. Notably, chronic administration of Ang 1-7 to CHF rats reduced cardiac hypertrophy. In conclusion, Ang 1-7 exerts beneficial renal and cardiac effects in rats with CHF. Thus, we postulate that ACE2/Ang 1-7 axis represents a compensatory response to over-activity of ACE/AngII/AT1R system characterizing CHF and suggest that Ang 1-7 may be a potential therapeutic agent in this disease state. Full article

Kidney injury molecule-1 (KIM-1) is a biomarker of renal injury and a predictor of cardiovascular disease. Aldosterone, via activation of the mineralocorticoid receptor, is linked to cardiac and renal injury. However, the impact of mineralocorticoid receptor activation and blockade on KIM-1 is uncertain. We investigated whether renal KIM-1 is increased in a cardiorenal injury model induced by L-NAME/ANG II, and whether mineralocorticoid receptor blockade prevents the increase in KIM-1. Since statin use is associated with lower aldosterone, we also investigated whether administering eiSther a lipophilic statin (simvastatin) or a hydrophilic statin (pravastatin) prevents the increase in renal KIM-1. Female Wistar rats (8–10 week old), consuming a high salt diet (1.6% Na+), were randomized to the following conditions for 14 days: control; L-NAME (0.2 mg/mL in drinking water)/ANG II (225 ug/kg/day on days 12–14); L-NAME/ANG II + eplerenone (100 mg/kg/day p.o.); L-NAME/ANG II + pravastatin (20 mg/kg/day p.o.); L-NAME/ANG II + simvastatin (20 mg/kg/day p.o.). Groups treated with L-NAME/ANG II had significantly higher blood pressure, plasma and urine aldosterone, cardiac injury/stroke composite score, and renal KIM-1 than the control group. Both eplerenone and simvastatin reduced 24-h urinary KIM-1 (p = 0.0046, p = 0.031, respectively) and renal KIM-1 immunostaining (p = 0.004, p = 0.037, respectively). Eplerenone also reduced renal KIM-1 mRNA expression (p = 0.012) and cardiac injury/stroke composite score (p = 0.04). Pravastatin did not affect these damage markers. The 24-h urinary KIM-1, renal KIM-1 immunostaining, and renal KIM-1 mRNA expression correlated with cardiac injury/stroke composite score (p < 0.0001, Spearman ranked correlation = 0.69, 0.66, 0.59, respectively). In conclusion, L-NAME/ANG II increases renal KIM-1 and both eplerenone and simvastatin blunt this increase in renal KIM-1. Full article

In Dahl salt-sensitive (SS) rats, impaired vascular relaxation can be restored by: (1) minipump infusion of a low (sub-pressor) dose of angiotensin II (ANG II) to restore physiological levels of plasma ANG II, (2) inhibition of 20-HETE production, and (3) introgression of a normally functioning renin allele from the Brown Norway rat (SS-13^BN consomic rat). Unlike SS rats, SS-13^BN rats have normal levels of ANG II on a normal-salt diet and suppressed ANG II on a high-salt (HS) diet. This study tested whether chronically low ANG II levels in SS rats upregulate cytochrome P450-4A (CYP4A) increasing the production of the vasoconstrictor 20-HETE. Although salt-induced suppression of ANG II levels increased reactive oxygen species (ROS) in basilar arteries from SS-13^BN rats in previous studies, this study showed no change in vascular 20-HETE levels in response to ANGII suppression. CYP4A inhibition significantly reduced vascular ROS levels and restored endothelium-dependent relaxation in response to acetylcholine in the middle cerebral artery (MCA) of SS rats and HS-fed SS-13^BN rats. These data demonstrate that both the renin–angiotensin system and the CYP4A/20-HETE pathway play a direct role in the vascular dysfunction of the Dahl SS rat but are independent of each other, even though they may both contribute to vascular dysfunction through ROS production. Full article

Hypertension is associated with increased expression of kinin B1 receptors (B1R) and increased levels of pro-inflammatory cytokines within the neurons. We previously reported that angiotensin II (Ang II) upregulates B1R expression and can induce neuroinflammation and oxidative stress in primary hypothalamic neurons. However, the order in which B1R activation, neuroinflammation, and oxidative stress occur has not yet been studied. Using primary hypothalamic neurons from neonatal mice, we show that tumor necrosis factor (TNF), lipopolysaccharides (LPS), and hydrogen peroxide (H₂O₂) can upregulate B1R expression and increase oxidative stress. Furthermore, our study shows that B1R blockade with R715, a specific B1R antagonist, can attenuate these effects. To further confirm our findings, we used a deoxycorticosterone acetate (DOCA)-salt model of hypertension to show that oxidative stress is upregulated in the hypothalamic paraventricular nucleus (PVN) of the brain. Together, these data provide novel evidence that relationship between oxidative stress, neuroinflammation, and B1R upregulation in the brain is bidirectional, and that B1R antagonism may have beneficial effects on neuroinflammation and oxidative stress in various disease pathologies. Full article

A high salt (HS) diet is associated with an increased risk for cardiovascular diseases (CVDs) and fibrosis is a key contributor to the organ dysfunction involved in CVDs. The activation of the renin angiotensin type 2 receptor (AT₂R) has been considered as organ protective in many CVDs. However, there are limited AT₂R-selective agonists available. Our first reported β-substituted angiotensin III peptide, β-Pro⁷-AngIII, showed high selectivity for the AT₂R. In the current study, we examine the potential anti-fibrotic and anti-inflammatory effects of this novel AT₂R-selective peptide on HS-induced organ damage. FVB/N mice fed with a 5% HS diet for 8 weeks developed cardiac and renal fibrosis and inflammation, which were associated with increased TGF-β1 levels in heart, kidney and plasma. Four weeks’ treatment (from weeks 5–8) with β-Pro⁷-AngIII inhibited the HS-induced cardiac and renal fibrosis and inflammation. These protective effects were accompanied by reduced local and systemic TGF-β1 as well as reduced cardiac myofibroblast differentiation. Importantly, the anti-fibrotic and anti-inflammatory effects caused by β-Pro⁷-AngIII were attenuated by the AT₂R antagonist PD123319. These results demonstrate, for the first time, the cardio- and reno-protective roles of the AT₂R-selective β-Pro⁷-AngIII, highlighting it as an important therapeutic that can target the AT₂R to treat end-organ damage. Full article

High and low sodium diets are associated with increased blood pressure and cardiovascular morbidity and mortality. The paradoxical response of elevated BP in low salt diets, aka inverse salt sensitivity (ISS), is an understudied vulnerable 11% of the adult population with yet undiscovered etiology. A linear relationship between the number of single nucleotide polymorphisms (SNPs) in the dopamine D₂ receptor (DRD2, rs6276 and 6277), and the sodium myo-inositol cotransporter 2 (SLC5A11, rs11074656), as well as decreased expression of these two genes in urine-derived renal proximal tubule cells (uRPTCs) isolated from clinical study participants suggest involvement of these cells in ISS. Insight into this newly discovered paradoxical response to sodium is found by incubating cells in low sodium (LS) conditions that unveil cell physiologic differences that are then reversed by mir-485-5p miRNA blocker transfection and bypassing the genetic defect by DRD2 re-expression. The renin-angiotensin system (RAS) is an important counter-regulatory mechanism to prevent hyponatremia under LS conditions. Oversensitive RAS under LS conditions could partially explain the increased mortality in ISS. Angiotensin-II (AngII, 10 nmol/L) increased sodium transport in uRPTCs to a greater extent in individuals with ISS than SR. Downstream signaling of AngII is verified by identifying lowered expression of nuclear factor erythroid 2-related factor 2 (NRF2), CCCTC-binding factor (CTCF), and manganese-dependent mitochondrial superoxide dismutase (SOD2) only in ISS-derived uRPTCs and not SR-derived uRPTCs when incubated in LS conditions. We conclude that DRD2 and SLC5A11 variants in ISS may cause an increased low sodium sensitivity to AngII and renal sodium reabsorption which can contribute to inverse salt-sensitive hypertension. Full article

Donors of nitroxyl and nitroxyl anion (HNO/NO⁻) are considered to be promising pharmacological treatments with a wide range of applications. Remarkable chemical properties allow nitroxyl to function as a classic antioxidant. We assume that HNO/NO⁻ can level down the non-enzymatic glycation of biomolecules. Since erythrocyte hemoglobin (Hb) is highly susceptible to non-enzymatic glycation, we studied the effect of a nitroxyl donor, Angeli’s salt, on Hb modification with methylglyoxal (MG) and organic peroxide―tert-butyl hydroperoxide (t-BOOH). Nitroxyl dose-dependently decreased the amount of protein carbonyls and advanced glycation end products (AGEs) that were formed in the case of Hb incubation with MG. Likewise, nitroxyl effectively protected Hb against oxidative modification with t-BOOH. It slowed down the destruction of heme, formation of carbonyl derivatives and inter-subunit cross-linking. The protective effect of nitroxyl on Hb in this system is primarily associated with nitrosylation of oxidized Hb and reduction of its ferryl form, which lowers the yield of free radical products. We suppose that the dual (antioxidant and antiglycation) effect of nitroxyl makes its application possible as part of an additional treatment strategy for oxidative and carbonyl stress-associated diseases. Full article