Rat Liver Membranes Research Articles

Effect of Talatisamine and its Derivate 14-O-Benzoyltalatisamine on Functional State of Rat Liver and Heart Mitochondria

Dysfunction of the mitochondria of various tissues causes the development of most pathological processes, including ischemia. In recent years, great attention has been paid to the use of plant biologically active substances in the prevention and treatment of pathological processes related to mitochondrial dysfunction. This is very relevant in relation to ischemic diseases and is of scientific and practical importance in the search for new pharmacological agents that correct the functions of damaged mitochondria for their treatment. The mitochondrial permeability transition pore (mPTP) actively participates in the regulation of mitochondrial functions, in the development of various pathological conditions and, at the same time, targets for various drugs and some biologically active substances. In vitro experiments evaluated the effects of alkaloids talatisamine and 14-O-benzoyltalatisamine on rat liver and heart Ca2+-dependent mPTP and lipid peroxidation (LPO) induced by Fe2+/ascorbate system. The investigated diterpenealkaloids inhibited the opening of the Ca2+-dependent mPTP in the membranes of rat liver and heart mitochondria. It was found that 14-O-benzoyltalatizamine inhibits the Ca2+-dependent conductance pore of rat liver and heart mitochondria more strongly than talatisamine. To compare the effects of 14-O-benzoyltalatisamine on rat liver and heart mPTP, concentrations from 1 μM to 200 μM were investigated. At these concentrations, liver mPTP was reliably inhibited by 10% to 81% and heart mPTP by 3.6% to 71.5% relative to control. The high sensitivity of diterpene alkaloids to the Ca2+-dependent permeability transition pore of liver mitochondria compared to heart mitochondria indicates their tissue specificity. The investigated alkaloids exhibited antioxidant properties by inhibiting Fe2+/ascorbate-induced mitochondrial suppression (LPO process) and MDA formation in membranes. LPO induced by Fe2+/ascorbate system in mitochondrial membranes was more actively inhibited by 14-O-benzoyltalatisamine. According to the results of the research, acylation of the hydroxyl group at the C-14 position of talatisamine by benzoyl chloride caused a rise in molecular activity of the derivative due to the introduction of the benzoyl group.

Design, development and bio-evaluation of a novel radio-ligand 99mTc-THQ-DTPA as a sigma 2 receptor specific breast tumor imaging agent

Over-expression of sigma-2 receptor in cancer cells provides an opportunity to develop molecular probes for diagnosis, even for non-receptor specific malignancies like triple negative breast cancers. In this work, a novel sigma-2 receptor ligand [THQ-DTPA] has been synthesized and characterized using 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (THQ) and diethylenetriaminepentaacetic acid (DTPA). The ligand is further chelated with 99mTc for application as metal based radiotracer [99mTc-THQ-DTPA]. Radiolabelling with 99mTc was achieved in an excellent yield of 98.0 ± 0.5% using stannous chloride as a reducing agent. The radioligand was found to be stable in human serum up-to 24 h, bio-compatible with less than 4% hemolysis, and exhibited high binding with sigma receptors isolated from rat liver membrane (Kd of 16.32 ± 4.93 nM and Bmax of 0.5232 ± 0.06 pmol/mg). Bio-distribution studies in triple-negative breast tumor bearing nude mice showed high tumor uptake after 30 min of injection with tumor/muscle (T/M) ratio of 3.58 ± 0.09. At 240 min, the T/M ratio (2.84 ± 0.20) decreased by 35% when administered in sigma blocked tumor bearing mice (1.81 ± 0.16) suggesting the selectivity of the ligand. Tumor imaging in gamma camera indicated a contrast of 3.56 at 30 min p.i. The above findings indicate that the ligand 99mTc-THQ-DTPA binds to sigma-2 receptors with high affinity and has potential for triple-negative breast tumor imaging.

Very Low Protein Diets Induce a Rapid Decrease in Hepatic cAMP-Dependent Protein Kinase Followed by a Slower Increase in Adenylyl Cyclase Activity in Rats

Recent evidence indicates that cAMP-mediated responses are desensitized in liver during malnutrition. While receptor-stimulated production of cAMP is increased in hepatocytes from rats fed very low protein diets for 14 d, activity of cAMP-dependent protein kinase (PKA) is decreased in liver cytosol. The present study investigated the time course for this desensitization. Weanling rats were fed either a 0.5 (malnourished) or 15% protein (control) diet for 1, 3, 7 or 14 d. Total PKA activity decreased after only 3 d of feeding the low protein diet. This decrease was confined to the cytosolic compartment and was associated with a lower quantity of immunoreactive RI regulatory subunit of PKA, with no difference in the quantity of immunoreactive RII regulatory subunit. In contrast, basal-, MnCl2- and guanine nucleotide regulatory protein-stimulated adenylyl cyclase activities were not greater in liver membranes of malnourished rats than in those of the control rats until the 2nd wk of feeding. Greater activity was paralleled by an increase in the quantity of the stimulatory guanine nucleotide regulatory protein at d 14. The inhibitory guanine nucleotide regulatory protein quantity did not differ between dietary groups. Greater cAMP production was not mediated by changes in PKA phosphorylation of adenylyl cyclase because preincubation of membranes with purified PKA catalytic subunit decreased MnCl2-stimulated cAMP production equally in liver membranes of both control and malnourished rats. Similarly, treatment with alkaline phosphatase decreased adenylyl cyclase activity but did not eliminate the difference in adenylyl cyclase activity between control and malnourished rats. These data demonstrate that loss of PKA activity is an early response to a low protein diet and that, subsequently, a number of molecular adaptations occur which increase cAMP production. These changes may be adaptive responses to malnutrition that maintain essential cAMP-dependent functions.

Comparative evaluation of biased agonists Sarcosine1 , d-Alanine8 -Angiotensin (Ang) II (SD Ang II) and Sarcosine1 , Isoleucine8 -Ang II (SI Ang II) and their radioiodinated congeners binding to rat liver membrane AT1 receptors.

Angiotensin II analogue and β-arrestin biased agonist TRV027 (Sarcosine1 , d-Alanine8 -Angiotensin (Ang) II; SD Ang II), developed by Trevena, Inc. in the early 2010s, brought hopes of a novel treatment for cardiovascular diseases, due to its ability to simultaneously cause signaling through the β-arrestin signaling pathway, while antagonizing the pathophysiological effects of Ang II mediated by the AT1 receptor G protein signaling cascades. However, a phase II clinical trial of this agent revealed no significant benefit compared to placebo treatment. Using 125 I-Sarcosine1 , Isoleucine8 -Ang II (125 I-SI Ang II) radioligand receptor competition binding assays, we assessed the relative affinity of TRV027 compared to SI Ang II for liver AT1 receptors. We also compared radioiodinated TRV027 (125 I-SD Ang II) binding affinity for liver AT1 receptors with 125 I-SI Ang II. We found that despite its anticipated gain in metabolic stability, TRV027 and 125 I-SD Ang II had reduced affinity for the AT1 receptor compared with SI Ang II and 125 I-SI Ang II. Additionally, male-female comparisons showed that females have a higher AT1 receptor density, potentially attributed to tissue-dependent estrogen and progesterone effects. Peptide drugs have become more popular over the years due to their increased bioavailability, fast onset of action, high specificity, and low toxicity. Even though Trevena®'s biased agonist peptide TRV027 offered greater stability and potency compared to earlier AT1 R biased agonists, it failed its phase II clinical trial in 2016. Further refinements to AT1 R biased agonist peptides to improve affinity, as seen with SI Ang II, with better stability and bioavailability, has the potential to achieve the anticipated biased agonism.

125I-Angiotensin 1-7 binds to a different site than angiotensin 1-7 in tissue membrane preparations.

To study the receptor for Angiotensin (Ang) 1-7 using a radioligand (125I-Ang 1-7)-binding assay. For more than a decade, Mas has been viewed as the receptor for Ang 1-7; however, Ang 1-7 binding has not been pharmacologically characterized in tissue membrane preparations. Radioligand-binding assays were carried out using tissue membrane preparations using radioiodinated Angiotensin 1-7 (125I-Ang 1-7) to characterize its binding site. Non-radioactive 127I-Ang 1-7 was used to test if the addition of an iodine to the tyrosine4 moiety of Ang 1-7 changes the ability of Ang 1-7 to competitively inhibit 125I-Ang 1-7 binding. 125I-Ang 1-7 binds saturably, with moderately high affinity (10-20 nM) to a binding site in rat liver membranes that is displaceable by 127I-Ang 1-7 at nanomolar concentrations (IC50 = 62 nM) while Ang 1-7 displaces at micromolar concentrations (IC50 = 80 µM) at ~22 °C. This binding was also displaceable by inhibitors of metalloproteases at room temperature. This suggests that 125I-Ang 1-7 binds to MMPs and/or ADAMs as well as other liver membrane elements at ~ 22 °C. However, when 125I-Ang 1-7-binding assays were run at 0-4 °C, the same MMP inhibitors did not effectively compete for 125I-Ang 1-7. The addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the binding characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors.

125‐I‐Sarcosine(1), D‐Alanine(8) Angiotensin II as a Biased Radioligand for AT1 Angiotensin II Receptors

The concept of biased agonism of G protein‐coupled receptors arose from studies of the AT1 subtype of angiotensin (Ang) II receptor. A putative antagonist ligand, sarcosine1, isoleucine4, isoleucine8 Ang II (SII) showed negligible ability to activate G protein signaling pathways of the AT1 receptor, but was able to cause AT1 receptor internalization and mitogen activated protein (MAP) kinase activation at micromolar concentrations. Subsequently, a different Ang II congener; sarcosine1, D‐alanine8 Ang II was developed as a potential therapeutic (TRV‐027; TRV120027) for treatment of acute heart failure. While clinical trials did not support a therapeutic benefit of sarcosine1, D‐alanine8 Ang II for treatment of heart failure, it still promises to be a better biased agonist than SII, which acts only at micromolar concentrations. To assess the potential use of sarcosine1, D‐alanine8 Ang II as a radioligand for quantification of Ang II receptor binding, we radiolabeled sarcosine1, D‐alanine8 Ang II with 125iodine and compared its binding to that of 125I‐sarcosine1 isoleucine8 Ang II in rat liver membranes, which express high levels of AT1 receptors and negligible levels of AT2 receptors. At concentrations up to 5.6 nM, 125I‐sarcosine1, D‐alanine8 Ang II showed little evidence of saturable binding to AT1 receptors, in contrast to 125I‐sarcosine1 isoleucine8 Ang II, which showed a KD of 0.6 nM. At 5.6 nM 125I‐sarcosine1 isoleucine8 Ang II binding to AT1 receptors was 8.5 times greater than 125I‐sarcosine1, D‐alanine8 Ang II. To verify that sarcosine1, D‐alanine8 Ang II binds to liver membrane AT1 receptors, we compared the ability of sarcosine1, D‐alanine8 Ang II and Ang II to compete for 125I‐sarcosine1 isoleucine8 Ang II binding. Sarcosine1 isoleucine8 Ang II and Ang II were similar in their ability to compete for AT1 receptor binding: 6.7±0.7 nM and 15.6±0.8 nM and 84.3±1.1 and 86.7±0.3 % of total binding (IC50, n=2±SD), respectively. This suggests that iodination of tyr4 of sarcosine1, D‐alanine8 Ang II alters its ability to bind to AT1 receptors and potentially act as a biased agonist. Continuing studies will examine the ability of higher concentrations of 125/127I‐sarcosine1, D‐alanine8 Ang II to interact with Ang II receptors.Support or Funding InformationNova Southeastern University President’s Faculty Research Development Grant, Cardiovascular Neuroscience Research Fund

Preliminary Characterization of the 125I‐Angiotensin 1‐7 Binding Site in Rodent Liver

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system that acts centrally and peripherally to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been viewed as the receptor for Ang 1‐7, however, Ang 1‐7 binding has not been pharmacologically characterized in tissue membrane preparations. In this study, we show that 125I‐Ang 1‐7 binds to a site for which Ang 1‐7 has low affinity. To assess the possibility that the low affinity of Ang 1‐7 to compete for 125/127I‐Ang 1‐7 binding was due to its susceptibility to metabolism by peptidases in the tissue membrane homogenate, the integrity of Ang 1‐7 as well as 127I‐Ang 1‐7 (iodoAng 1‐7) was evaluated by HPLC analysis. Both Ang 1‐7 and iodoAng 1‐7 were substantially metabolized in liver membrane homogenates at room temperature (RT). However, when incubated on ice (~0 C), 125I‐Ang 1‐7 binds with moderately higher affinity (~3 nM versus 10–20 nM at RT) to a binding site in rat liver membranes that is displaceable by iodoAng 1‐7 at nanomolar concentrations, but Ang 1‐7 still shows a low affinity to compete (1.4μM for Ang 1‐7and 69.4nM for iodoAng 1‐7). At RT 125I‐Ang 1‐7 binding is partially displaceable by metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors, e.g., marimastat, batimastat, and GM‐6001 at nanomolar IC50 values, similar to their IC50 values for MMP activity, suggesting that 125I‐Ang 1‐7 binds to MMPs and/or ADAMs as well as other tissue elements at RT. However, when incubated at ~0 C, the MMP and ADAM inhibitors had micromolar IC50 values, suggesting that 125I‐Ang 1‐7 binding to these peptidases is temperature dependent. Our results continue to suggest that the addition of an iodine molecule to the tyrosine in position 4 of Ang 1‐7 drastically changes the characteristics of this peptide making it unsuitable for characterization of Ang 1‐7 receptors. Continuing studies are underway to identify the peptidase(s) or other binding moieties to which 125I‐Ang 1‐7 binds to determine their potential therapeutic importance as druggable targets.

Distinct Regulation of σ 1 Receptor Multimerization by Its Agonists and Antagonists in Transfected Cells and Rat Liver Membranes.

Extensive studies have shown that the σ 1 receptor (σ 1R) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of σ 1R as a homotrimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates σ 1R oligomerization. Here, novel nondenaturing gel methods and mutational analysis were used to examine σ 1R oligomerization. In transfected cells, σ 1R exhibited as multimers, dimers, and monomers. Overall, σ 1R agonists decreased, whereas σ 1R antagonists increased σ 1R multimers, suggesting that agonists and antagonists differentially affect the stability of σ 1R multimers. Endogenous σ 1R in rat liver membranes also showed similar regulation of oligomerization as in cells. Mutations at key residues lining the trimerization interface (Arg119, Asp195, Phe191, Trp136, and Gly91) abolished multimerization without disrupting dimerization. Intriguingly, truncation of the N terminus reduced σ 1R to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of σ 1R. The E102Q σ 1R mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of σ 1R multimeric assembly may impair its function. Interestingly, oligomerization of σ 1R was pH-dependent and correlated with changes in [3H](+)-pentazocine binding affinity and Bmax Combined with mutational analysis, it is reasoned that σ 1R multimers possess high-affinity and high-capacity [3H](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that σ 1R may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand-regulated σ 1R multimerization may provide novel approaches to modulate the function of σ 1R and its interacting proteins. SIGNIFICANCE STATEMENT: The σ 1 receptor (σ 1R) modulates the activities of various partner proteins. Recently, crystal structures of σ 1R were elucidated as homotrimers. This study used novel nondenaturing gel methods to examine σ1R oligomerization in transfected cells and rat liver membranes. Overall, agonist binding decreased, whereas antagonist binding increased σ 1R multimers, which comprised trimers and larger units. σ 1R multimers were shown to bind [3H](+)-pentazocine with high affinity and high capacity. Furthermore, mutational analysis revealed a crucial role of its N-terminal domain in σ 1R multimerization.

Abstract P2027: Preliminary Characterization Of The 125 I-angiotensin 1-7 Binding Site In Rat Liver

Angiotensin 1-7 (Ang 1-7) is a product of the renin-angiotensin system (RAS) that can reduce blood pressure and fibrosis. For more than a decade Mas has been viewed as the receptor for Ang 1-7, however, there are lingering doubts regarding the nature of the interaction between the heptapeptide and Mas. To date, no one has pharmacologically characterized Ang 1-7 binding in tissue membrane preparations. In this study we show that 125 I-Ang 1-7 binds to a site for which Ang 1-7 has low (μmolar) affinity, suggesting that the addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors. 125 I-Ang 1-7 binds with moderately high affinity (10-20 nM) and saturability to a binding site in rat liver membranes that is displaceable by 127 I-Ang 1-7 (iodoAng 1-7) at nanomolar concentrations. This binding is also displaceable by inhibitors of metalloproteases. To test the hypothesis that 125 I-Ang 1-7 binds to a peptidase, a series of peptidase inhibitors were evaluated for their ability to inhibit 125 I-Ang 1-7 binding. The non-selective matrix metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors marimastat, batimastat, and GM-6001, which inhibit MMP1,2,3,7,8,13,14 and ADAM 9,17, partially inhibited 125 I-Ang 1-7 binding with nanomolar affinities, similar to their IC 50 values for MMPs, suggesting that 125 I-Ang 1-7 binds to MMPs and/or ADAMs as well as other tissue elements. Further testing with the specific MMP-2 (ARP100), MMP-9 and ADAM-17 inhibitors indicate that 125 I-Ang 1-7 is not binding to these peptidases. Continuing studies are underway to establish conditions under which the 125 I-Ang 1-7 ligand is not metabolized by peptidases, while retaining the ability to bind to peptidases is underway to enable the identification of the specific peptidase(s) or other binding moieties to which 125 I-Ang 1-7 binds to determine their potential therapeutic importance as a druggable targets.

Abstract 1294: Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A

Abstract Sigma-2 receptors, recently identified as TMEM97, are highly expressed in various types of cancer cells. Sigma-2 receptor agonists have traditionally been characterized as ligands that induce programmed cell death in various cancer cell types. Sigma-2 receptor-mediated cell death involves a number of mechanisms including caspase activation, mitochondrial depolarization, and autophagy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor with stimulation of glycolytic hallmarks, including increase in cellular ATP level, reduced ROS, and stabilization of HIF-1α. We have investigated a family of 6-substituted analogs of the canonical sigma-2 receptor antagonist, SN79, members of which can produce both the metabolic stimulative and the cytotoxic effects. Here we compare the activities of two closely related compounds. CM571, the 6-amino derivative, bound with high affinity to both sigma-1 and sigma-2 receptors (sigma-1 Ki = 15.5 nM; sigma-2 Ki = 21.7 nM). MAM03055A (MAM) is essentially a homo-bivalent dimer of CM571, linked by a thiourea moiety. MAM exhibited high affinity and selectivity for sigma-2 receptors (sigma-1 Ki = 3,371 nM; sigma-2 Ki = 55.9 nM). The functional effects of the two compounds were compared in human SK-N-SH neuroblastoma cells. CM571 caused a dose-dependent stimulation of MTT reduction, indicating the metabolic stimulative-effect, producing 40% stimulation at 30 µM. By contrast, MAM induced dose-dependent cell death with an EC50 = 4.24 µM. MAM induced time-dependent cleavage of proapoptotic Bid, a key component of the programmed cell death mechanism of sigma-2 ligands in these cells. Previous studies had shown that only irreversibly binding, isothiocyanate analogs of SN79 induced programmed cell death, while other analogs produced metabolic stimulation. Rat liver membranes were treated with compound, washed to remove unbound ligand, and then subjected to radioligand binding to determine recovery of sigma-1 receptors ([3H](+)-pentazocine) and sigma-2 receptors ([3H]DTG + (+)-pentazocine). Treatment with CM571 resulted in full recovery of both sigma-1 and sigma-2 binding. However, MAM treatment resulted in loss of sigma-2 binding and full recovery of sigma-1 activity, indicating irreversible binding to the sigma-2 receptor. Since there is no chemically reactive moiety in MAM to react covalently, MAM binds “pseudo-irreversibly”. Treatment of cells with MAM for 60 min, followed by washout produced nearly comparable levels of cell death 24 h later as continuous exposure of cells for 24 hours, consistent with pseudo-irreversible action at the sigma-2 receptor. The results show that mono-valent and bivalent sigma-2 ligands in this series show different modes of interaction with the receptor, resulting in divergent effects on cell viability. Citation Format: Cheri Z. Liu, Bridget M. McVeigh, Marco Mottinelli, Hilary E. Nicholson, Christopher R. McCurdy, Wayne D. Bowen. Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1294.

Development of a Novel Radioligand for Characterization of the Mas Receptor for Angiotensin 1–7

It is well established that Mas (NP_002368) is the receptor for angiotensin 1–7 (Ang 1–7), however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has repeatedly failed to reliably demonstrate high affinity, saturable binding of 125I‐Ang 1–7 in liver, kidney, brain and testes membrane preparations from both rats and mice. While the presence of a single iodine‐125 molecule on the tyrosine of Ang II does not impair its binding to the AT1 and AT2 receptors, it is not known whether the presence of an iodine‐125 molecule on the tyrosine of Ang 1–7 is equally innocuous. To begin to address this question, we prepared a novel analog of Ang 1–7: Tyr0‐Ang 1–7, to provide an alternate site for radioiodination of Ang 1–7. Tyr0‐Ang 1–7 was radioiodinated using the chloramine T procedure of Hunter and Greenwood (1962) with a 7 times excess of peptide to iodine to minimize di‐iodination of the peptide. Two radioiodinated peaks, presumed to be mono125I‐Y0‐Ang 1–7 and mono125I‐Y4‐Ang 1–7, were resolved from the uniodinated peptide peak by HPLC (C18 reverse phase column, 14.5% acetonitrile: triethylamine phosphate, pH 3.0 isocratic mobile phase). Saturation and competition radioligand binding assays were run for each peak using rat liver membrane preparations in the presence and absence of 10 μM Ang 1–7. While the identity of the early and late 125I‐Y0‐ Ang 1–7 peaks has not yet been established, preliminary studies indicate that both 125I‐Y0‐Ang 1–7 peaks displayed high affinity, saturable binding to rat liver membranes: early peak: KD = 7.7±2.3 nM, BMAX = 3.3±0.72 fmol/mg wet weight; and late peak KD = 11±2, BMAX = 3.6±0.4 fmol/mg wet weight. Competition binding assays run using the late peak 125I‐Y0‐Ang 1–7 revealed IC50's of 32±15 nM for Ang 1–7 and 15 ± 9.6 nM for Y0‐Ang 1–7. Comparison of the ability of a variety of Ang peptides to compete for late peak 125I‐Y0‐Ang 1–7 binding at a 10 μM concentration gave a rank order of Ang 1–7>Ang 3–7>Ang IV>Ang III>Ang 2–7>Ang II>Ang I. These results suggest that addition of a tyrosine at the amino terminus of Ang 1–7 may increase its binding affinity for Mas and that the presence of an iodine‐125 on either Y0 or Y4 of Ang 1–7 does not preclude high affinity saturable binding to Mas.Support or Funding InformationThis work was supported by the Cardiovascular Neuroscience Fund, Nova Southeastern University and the Peptide Radioiodination Shared Resource, Georgetown University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of a high-fat diet on superoxide anion generation and membrane fluidity in liver mitochondria in rats

BackgroundObesity is a primary factor of lifestyle-related diseases, and the age of its onset has decreased. The reactive oxygen species (ROS), the superoxide anion, is generated in the mitochondrial electron transport chain and the damage it induces in cells may be a contributing factor to obesity-related lifestyle diseases. In the present study, the influence of the ingestion of a high-fat diet (HFD) on superoxide anion generation in rat liver mitochondria (Mt) and membrane fluidity was investigated.MethodsMale Wistar rats were fed a normal diet (ND, n = 6) or HFD (n = 6). Liver Mt were isolated and oxygen consumption, superoxide anion production (the adrenaline method), and membrane fluidity (the spin label method) were measured.ResultsAfter 11 weeks, body weights and abdominal circumferences were higher in the HFD group than in the ND group. Mt oxygen consumption was higher in the HFD group than in the ND group. Superoxide anion production was significantly lower in the HFD group than in the ND group, while no significant changes were observed in membrane fluidity.ConclusionAlthough rats developed diet-induced obesity, it did not reach the level of disease development. The promotion of lipid metabolism appeared to reduce superoxide anion production, but did not influence membrane fluidity. While superoxide anion damages cells as an oxidative stress, ROS and superoxide dismutase are essential signaling molecules in the body. The present results suggest that the continuous ingestion of a HFD impairs Mt and induces disease development.

Terlipressin, a vasoactive prodrug recommended in hepatorenal syndrome, is an agonist of human V1, V2 and V1B receptors: Implications for its safety profile

Terlipressin is recommended as a gold standard to treat hepatorenal syndrome complicating liver cirrhosis. It is presented as a specific V1A receptor agonist, beyond its enzymatic conversion into lysine8-Vasopressin (LVP), able to counteract the splanchnic vasodilation. However, the complete pharmacological characterization of this drug with respect to the different vasopressin receptor subtypes is missing. We studied terlipressin intrinsic properties, focusing not only on V1A, but also on other vasopressin receptor subtypes. The experimental studies were conducted on rat and human cellular models. Binding experiments were performed on rat liver membranes and CHO cells transfected with the different human vasopressin receptor subtypes. Agonist status was assessed from inositol phosphate or cyclic AMP assays, and measurement of intracellular calcium variations, performed on cultured vascular smooth muscle cells from rat aorta and human uterine artery and CHO cells. Terlipressin binds to the rat and human V1A receptors with an affinity in the micromolar range, a value 120 fold lower than that of LVP. It induces a rapid and transient intracellular calcium increase, a robust stimulation of phospholipase C but with reduced maximal efficiencies as compared to LVP, indicating a partial V1A agonist property. In addition, terlipressin is also a full agonist of human V2 and V1B receptors, with also a micromomolar affinity. ConclusionsTerlipressin is a non-selective vasopressin analogue, exhibiting intrinsic agonist properties. Its full V2 receptor agonism may result in renal effects potentially aggravating water retention and hyponatremia of cirrhosis.

Characterization of 125‐I‐Angiotensin (1–7) Binding to Mouse Forebrain and Liver and Rat Liver

Angiotensin II (Ang II) is a potent regulator of blood pressure and is pathogenetic for cardiovascular disease. Identification of the ACE homologue ACE2, which forms Ang 1–7 from Ang II, and the GPCR Mas as a functional Ang 1–7 receptor has provided biochemical and molecular tools to study the role of Ang 1–7 as a counter regulator of the pathogenesis of Ang II. Despite the physiological importance of Ang1–7, no one has demonstrated saturable 125I‐Ang 1–7 binding in mammalian tissue. To date, only single concentration, specific binding of 125I‐Ang 1–7 to mouse kidney has been reported. To assess the saturability of 125I‐Ang 1–7 binding to mammalian tissue, we prepared a low specific activity 125/127I‐Ang 1–7 by radioiodinating Ang 1–7 with 1 part 125I:19 parts 127I, using chloramine T at pH 7.5 to limit radioiodination to tyr4, and pH 8.5 to radioiodinate both tyr4 and his6, with subsequent purification of monoradioiodinated Ang 1–7 using reverse‐phase HPLC. Forebrains from adult male athymic mice (nu/+) were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:EDTA (50:5 mM) pH 7.3 and incubated with 12 concentrations of 125/127I‐Ang 1–7 ranging from 3–110 nM for 30 min at 22 C, after which bound 125/127I‐Ang 1–7 was collected on GF/B filters and counted. Both radio ligands showed specific (100 μM Ang 1–7 displaceable) saturable binding: 35±8.3 and 35±4.2 fmol/mg initial wet weight (mean±SEM) with KD = 42.6±19.2 and 47.9±13.9 nM, for pH 7.5 and pH 8.5 preparations, respectively. This suggests that the presence of iodine on tyr4 or tyr4 + his6 does not differentially affect binding of Ang 1–7 to mouse brain membranes. Preliminary studies indicate that Ang II, Ang IV, Ang 3–7, Ang 4–7, Sar1, Ile8 Ang II, losartan and PD123319 have low affinity for the 125/127I‐Ang 1–7 binding site in mouse brain membranes. 125/127I‐Ang 1–7 (prepared at pH 8.5) binding to mouse liver using 50 mM Tris: 5 mM MgCl2, pH 7.2, also was saturable 179±10 fmol/g with KD = 20.4±4 nM. 125/127I‐Ang 1–7 (prepared at pH 7.5) binding to rat liver membranes using 50 mM Tris: 5 mM MgCl2 pH 7.2 also was saturable 66.7±1.8 fmol/g with KD = 7.1±1.7 nM. No saturable binding was observed in rat heart. Future studies will assess whether this binding is to the mas protein using other rodent tissues, e.g., kidney, as well as mas knockout mice. If these studies document that 125/127I‐Ang 1–7 binds to mas, this radio ligand can be used to assess the binding functionality of mas as well as to measure differences in the binding functionality of mas in animal models of cardiovascular disease.Support or Funding InformationF.F. Conti: CAPES: 99999.008874/2014‐00. A. Linares: None. L. Couling: None. M. Morris: DOD grant W81XWH‐13‐2‐0085. K. De Angelis: None. R.C. Speth: Research Grant; Significant; NIH HL‐113905; Pilot Award from the Translational Technologies Component of the Georgetown, Howard Universities Center for Clinical and Translational Science [UL1TR000101]. Other Research Support; Significant; Cardiovascular Neuroscience Research Fund, Nova Southeastern University.

Identification of the Sigma-2 Receptor: Distinct from the Progesterone Receptor Membrane Component 1 (PGRMC1).

σRs are unique intracellular chaperone proteins [8] initially thought to be opioid receptor subtypes [9]. They have been classified into two subtypes based on specific radioligand binding assays using [3H](+)pentazocine for σ1Rs and [3H]1,3-di-o-tolylguanidine ([3H]DTG, in the presence of dextrallorphan to mask the σ1R) for σ2Rs in rat liver and kidney membranes [10]. Currently, the more selective σ1R ligand (+)-pentazocine has replaced dextrallorphan to mask the σ1R [7,11-14]. The σ1R has already been cloned as a 25-29 kDa chaperone protein composed of 223 amino acids [4,8,15]. It is widely distributed throughout the body [16-20]. Upon binding with agonists or under cellular stress, σ1Rs translocate from their primary endoplasmic reticulum (ER) location to different subcellular compartments where they can regulate ion channels and G-protein-coupled-receptor (GPCR) signaling [8,21-24]. In vivo functional studies on σ1Rs suggest that they play a substantial role in various cellular functions. Drugs acting at this receptor have been studied for their potential therapeutic effects in cancer, human immunodeficiency virus (HIV) infection, various psychiatric disorders, and substance abuse [1,25].

Improvement of a sample preparation method assisted by sodium deoxycholate for mass-spectrometry-based shotgun membrane proteomics†

In current shotgun-proteomics-based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Abstract 2242: Development of selective irreversible antagonists for sigma-2 receptors.

Abstract Sigma-1 and sigma-2 receptors are potential targets for therapeutic and diagnostic agents for various types of cancer. Sigma-1 receptors promote proliferation and survival, whereas activation of sigma-2 receptors induces apoptosis. Thus, there has been particular focus on sigma-2 receptors, which may be related to the PGRMC1 complex. While several selective ligands for sigma-2 receptors have been developed, there is a scarcity of tools for studying structure and function, particularly antagonists. SN-79 is a high affinity ligand for sigma-2 receptors, with 10-fold selectivity for sigma-2 over sigma-1 (Ki = 30 nM vs. 290 nM, respectively). Four isothiocyanate derivatives of SN-79 were characterized in rat liver membranes using [3H](+)-pentazocine to label sigma-1 and [3H]DTG (in presence of unlabeled (+)-pentazocine) for sigma-2. The isothiocyanate derivatives all exhibited lower affinity for sigma-2 receptors compared to SN-79. Sigma-2 Ki values were 320, 102, 395, and 271 nM for CM-572, CM-617, CM-621, and CM-769, respectively. Irreversible binding was examined by pretreating membranes for 60 min with the compound, extensively washing the membranes, and then determining recovery of radioligand binding. Loss of radioligand binding upon pretreatment results from “wash-resistant binding” of the compound and indicates covalent attachment to the receptor. Pretreatment with 100 nM CM-572 or CM-617 resulted in recovery of only 25% and 40% of sigma-2 binding, respectively. Treatment with 100 nM CM-621 or CM-769 had no effect. Therefore, while all four compounds bind to sigma-2 receptors with roughly comparable affinities, only CM-572 and CM-617 bind irreversibly. None of the compounds had an effect on sigma-1 receptor binding, indicating selective acylation of the sigma-2 receptor. In dose studies, CM-572 exhibited an IC50 ∼30 nM. Human SK-N-SH neuroblastoma cells were used to examine the biological activity of CM-572. Cells were pretreated for 1 h with CM-572 at concentrations up to 100 uM, then washed free of ligand, normal media added, and monitored by MTT assay after 24 h. There was no effect on cell viability up to a concentration of 10 uM, where ∼20% cytotoxicity was observed. About 80% cytotoxicity was observed at 100 uM. This suggests that CM-572 may have an irreversible partial agonist effect. However, pretreatment with CM-572 was unable to attenuate cell death induced by the sigma-2 agonist, CB-64D. A component of the signaling cascade of sigma-2 receptor activation is calcium release from the ER. In Fura-2 loaded cells, CM-572 produced no calcium signal at 3 uM and a small signal at 10 uM, though much smaller than the robust signal induced by CB-64D. Larger signals were produced at 30 uM and 100 uM CM-572. Interestingly, 3 uM and 10 uM CM-572 attenuated the calcium signal induced by 30 uM CB-64D. Taken together, these data indicate that CM-572 may be an irreversible partial agonist at sigma-2 receptors. Citation Format: Hilary Nicholson, Anthony Comeau, Christophe Mesangeau, Christopher R. McCurdy, Wayne D. Bowen. Development of selective irreversible antagonists for sigma-2 receptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2242. doi:10.1158/1538-7445.AM2013-2242

Effect of sweet grass (Hierochloe odorata) on the physico-chemical properties of liver cell membranes from rats intoxicated with ethanol

Changes in the composition and physicochemical properties of liver cell membranes due to ethanol intoxication are due mainly to reactive oxygen species (ROS). The destructive action of free radicals can be neutralized by administration of antioxidants. The purpose of this study was to investigate the efficacy of sweet grass on the physicochemical and biochemical properties of the rat liver membrane altered by chronic ethanol intoxication. Qualitative and quantitative composition of phospholipids and proteins in the membrane were determined by HPLC. Ethanol increased phospholipid levels and altered the level of integral proteins as determined by decreased phenylalanine, cysteine and lysine. Ethanol significantly enhanced changes in the surface charge density of the liver cell membranes as determined by electrophoresis. Administration of sweet grass to rats intoxicated with ethanol significantly protects lipids and proteins against oxidative modifications. Therefore, sweet grass protects against some of the deleterious membrane changes associated with ethanol exposure.

Development and evaluation of an entirely solution-based combinative sample preparation method for membrane proteomics

The hydrophobic nature of many membrane proteins, especially integral membrane proteins, brings great difficulties to their analysis. To improve the analysis of membrane proteins, an entirely solution-based combinative sample preparation (CSP) method was developed and its application to the shotgun analysis of rat liver membrane proteomes was evaluated in this study. This CSP method comprehensively uses the strong ability of sodium dodecyl sulfate (SDS) to lyse the membranes and solubilize hydrophobic membrane proteins, the high efficiency of the optimized acetone precipitation method in sample cleanup and protein recovery, and the advantages of sodium deoxycholate (SDC) in improving protein solubilization/digestion as well as being compatible with trypsin activity. Compared with two other representative sample preparation methods, the SDC-assisted membrane-lysing method and the tube gel method, the newly established CSP method exhibited superiority in the recovery and identification of hydrophobic peptides, larger peptides, and highly hydrophobic membrane proteins with multiple transmembrane domains. The CSP method has characteristics of easy operation, low cost, and suitability for treating protein samples in various volumes, particularly large volumes, thereby having potential in the analysis of membrane proteomes with mass spectrometry.