bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosinergic Modulation Of The Imidazoline I1‐Receptor‐Dependent Hypotensive Effect Of Ethanol In Acute Renal Failure
Authors: El‐Mas Mahmoud M , El‐Gowelli Hanan M. , Harraz Osama F. , Ghazal Abdel‐Rheem M. , Mohy El‐Din Mahmoud M.
Published: June 2021

We recently reported that ethanol lowers blood pressure (BP) in rats with acute renal failure (ARF) via facilitation of sympathoinhibitory pathways of central imidazoline I1‐receptors. This study investigated whether adenosine receptor subtypes modulate the ethanol‐I1‐receptor interaction. The effect of selective blockade of adenosine A1, A2A, or A2B receptors on hemodynamic responses to consecutive administration of moxonidine, I1‐receptor agonist, and ethanol was determined in the glycerol model (10 ml/kg, i.m.) of ARF. Ethanol (1 g/kg i.v.) elicited significant decreases and increases in BP and heart rate (HR), respectively, in ARF rats. Prior exposure to moxonidine abolished the hypotensive but not the tachycardic effect of ethanol. The ability of moxonidine to abolish ethanol hypotension disappeared after selective blockade of adenosine A1 or A2A receptors with 8‐Cyclopentyl‐1,3‐dipropylxanthine and 8‐(3‐chlorostyryl) caffeine, respectively. In contrast, the moxonidine‐ethanol interaction was not affected in presence of alloxazine, a selective A2B antagonist or 8‐phenyltheophylline, a nonselective adenosine receptor antagonist. These findings suggest that alterations caused by moxonidine in adenosine A1 and A2A receptor signaling function to offset the I1‐receptor‐dependent hypotensive action of ethanol in ARF rats.
Aldehyde Dehydrogenase 1 in the Metabolism and Bioactivation of Organic Nitrates
Authors: Page Nathaniel A. , Tsou Pei‐Suen , Fung Sun Mi , Fung Ho‐Leung
Published: June 2021

The role of the cysteine residues, at position 302 and 303, of Aldehyde Dehydrogenase 1 (ALDH1) in the metabolism and activation of organic nitrates (ORN) was examined by selective mutation of these amino acids to alanine. C303A was devoid of dehydrogenase activity against acetaldehyde, while the C302A mutant exhibited slightly increased activity vs. the wild type, WT, (1.83 ± 0.61 vs 0.984 ± 0.115 U/mg, p<0.01). Cumulative nitric oxide (NO) generation was monitored via a NO electrode and NTG denitration was assessed via LCMS. ALDH1 C303A did not metabolize NTG significantly and failed to produce measurable NO. In comparison, C302A metabolism of NTG to its 1,2‐dinitrate metabolite was significantly slower than WT (p<0.05). NO production by C302A was decreased vs. WT with two model ORN, viz., isosorbide‐5‐mononitrate (5‐ISMN, 36.8 ± 6.6 % vs. WT, p<0.001) and NTG (45.1 ± 5.1 % vs. WT, p<0.01). Pre‐incubation with 0.1mM NTG, but not 1mM 5‐ISMN, for 10min significantly reduced WT ALDH1 dehydrogenase activity (7.07 ± 1.54 % vs control, p<0.001). C302A showed a similar pattern of dehydrogenase inhibition. ALDH1 C302A maintained the ability to metabolize 5‐ISMN which however is a poor substrate for ALDH2. These data suggest that C303 of ALDH1 is critical to its dehydrogenase, denitrating and NO generating activities against ORN while C302 participates to modulate these activities. (Supported in part by NIH grant HL81580)
Modulation of myocardial mitochondrial VDAC/MitoKATP channels in sepsis
Authors: Sharma Avadhesh C , Das Padmalaya , Chopra Mani
Published: June 2021

We hypothesized that modulation of myocardial voltage‐dependent anion channel (VDAC)/mitoKATP channels can affect the survival, release of cytochrome C during sepsis. Male Sprague‐Dawley rats (350–400g) were made septic using 200mg/kg cecal inoculum, ip. VDAC mRNA and protein expression was determined in the myocardium and isolated single adult rat ventricular myocytes (ARVMs) obtained from sham and septic rats. In addition, mitochondria and cytosolic fractions were isolated from ARVM treated with norepinephrine (NE, 10μmoles) ± 5‐Hydroxydecanoic acid (5HD; 100μmoles). The 5HD treatment at the time of the induction of sepsis increased the survival and reversed the sepsis‐induced hypotension and hypothermia at 6–24h. Sepsis‐induced mitochondrial membrane potential (ΔΨm) collapse in ARVMs was reversed by 5HD treatment. Sepsis also downregulated the colocalization of ANT with VDAC1 in isolated purified mitochondrial preparation (PMPs) obtained from septic ARVMs. Sepsis produced a significant down‐regulation of VDAC mRNA and protein along with upregulation of Bax in both myocardium and ARVMs. The 5HD treatment blocked NE‐induced decrease in VDAC expression and the release of cytochrome C in septic ARVMs. It appears that modulation of mitoKATP channels and increases the release of cytochrome C may play a crucial role in sepsis‐induced cardiomyocyte dysfunction.
TRP channel activation promotes dissociation of ET‐1/ETA‐complexes and terminates vasoconstriction in rat resistance arteries.
Authors: Meens Merlijn JPMT , Compeer Matthijs G , van Zandvoort Marc A , Hackeng Tilman M , De Mey Jo GR
Published: June 2021

Objectives

We tested whether TRP channel activation promotes dissociation of ET‐1/ETA‐complexes.

Methods

2nd order rat mesenteric arteries were investigated by two‐photon laser scanning microscopy and wire‐myography.

Results

ET‐1 (1–16nM) and rhodamine‐labeled ET‐1 (Rh‐ET‐1; 1–16nM) cause ETA‐mediated contractions that fade slowly after agonist removal and are insensitive to ETB‐antagonism. Contractions remaining after ET‐1 exposure are transiently reversed by several vasodilators, but persistently inhibited by i) TRP‐channel activators such as rutaecarpine (10 μM), allyl isothiocyanate (10 μM) and capsaicin (1 μM) or ii) CGRP (0.1 μM). CGRP‐receptor antagonists reduce the effects of TRP channel activators and CGRP. ET‐1 causes contractions with its original potency after reversal of ET‐1‐induced contractions by CGRP. Rh‐ET‐1 (16nM) binds to the tunica media of intact arteries. This persists after label removal but is abolished by CGRP (0.1 μM).

Conclusion

In arterial smooth muscle, ET‐1 binds “irreversibly” to ETA‐receptors; this causes prolonged signaling of the ET‐1/ETA‐complex. ET‐1/ETA‐complexes are a) insensitive to vasodilators or removal of free ligand and b) susceptible to allosteric inhibition by endogenous CGRP released by TRP channel activation.

This research was performed within the framework of project T2‐108 of the Dutch Top Institute Pharma.
A negative regulatory pathway of GLUT4 trafficking in adipocyte: new function of RIP140 in the cytoplasm via AS160
Authors: Ho Ping‐Chih , Lin Yi‐Wei , Tsui Yao‐Chen , Gupta Pawan , Wei Li‐Na
Published: June 2021

Receptor Interacting Protein 140 (RIP140), a nuclear receptor corepressor, is important for lipid and glucose metabolism. In adipocytes, RIP140 can be phosphorylated by protein kinase C epsilon, followed by arginine methylation, and exported to the cytoplasm. This study demonstrates for the first time a cytoplasmic function for RIP140: to counteract insulin‐stimulated glucose transporter 4 (GLUT4) membrane partitioning and glucose uptake in adipocytes. Cytoplasmic RIP140 interacts with the Akt substrate AS160, thereby impeding AS160 phosphorylation by Akt; this in turn reduces GLUT4 trafficking. This signal transduction pathway can be recapitulated in the epididymal adipocytes of diet‐induced obese mice: nuclear PKC epsilon is activated, cytoplasmic RIP140 increases, and GLUT4 trafficking and glucose uptake are reduced. The data reveal a new, cytoplasmic, function for RIP140 as a negative regulator of GLUT4 trafficking and glucose uptake, and shed insight into the regulation of basal and insulin‐stimulated glucose disposal by a nuclear‐initiated counteracting mechanism.

NIH grants DK54733, DK60521, DA11190 and K02‐DA13926 to L.‐N.W.
Activation of P2X7 receptor can lead to NAD+ release from astrocytes
Authors: Ying Weihai , Xia Weiliang , Chen Heyu
Published: June 2021

NAD+ plays critical roles in various biological processes. Recent studies have also suggested that extracellular NAD+ can induce death of a certain population of T cells, while there has been no information regarding the pathways by which intracellular NAD+ can be released into extracellular space. In this study we tested our hypothesis that activation of P2X7 receptors (P2X7R) can lead to NAD+ release from astrocytes. We found that the selective P2X7R agonist bzATP produced dose‐dependent decreases in intracellular NAD+ as well as increases in extracellular NAD+. The BzATP‐induced NAD+ loss was significantly attenuated by the P2 receptor antagonists PPADS and suramin, and completely blocked by the P2X7R antagonist oxATP. RNA silencing‐produced P2X7R reductions also led to prevention of the BzATP‐induced NAD+ release. Collectively, our results provide the first evidence demonstrating that NAD+ can be released from activated P2X7R, at least in such cell types as astrocytes (Supported by a Key Research Grant of Shanghai Municipal Scientific Committee, Pujiang Scholar Program Grant, Shanghai Engineering Center Grant of Equipment and Technology of Physical Therapy for Major Diseases, and a Shanghai Rising‐Star Program Grant).
Cytoplasmic RIP140 negatively modulates adiponectin secretion in adipocytes
Authors: Chuang Ya‐Shan , Ho Ping‐Chih , Persuad Shawna Devi , Lin Yi‐Wei , Chan Siu Chiu , Luo Sheng‐Ching , Wei Li‐Na
Published: June 2021

Receptor‐interacting protein 140 (RIP140) is known as a nuclear co‐repressor for various nuclear receptors. Recently, we identified a PKC epsilon‐triggered post‐translational modification pathway promotes RIP140 cytoplasmic accumulation which in turn blocks GLUT4 trafficking in adipocytes. Here we further examined the roles of cytoplasmic RIP140 in modulating adipokine secretion in adipocytes. We found that silencing RIP140 or PKC epsilon up‐regulated adiponectin secretion but did not affect its mRNA levels. Conditional medium from RIP140‐ or PKC epsilon‐knockdown 3T3‐L1 adipocytes increased glucose uptake in muscle cells as well as decreased glucose production in hepatocytes by promoting AMPK phosphorylation. However, adiponectin neutralized antibody can reverse conditional medium's effect on glucose uptake in muscle cells and glucose production in hepatocytes. Besides, we identified that endothelin‐1 (ET1) enhanced nuclear PKC epsilon activity resulted in RIP140 export from nucleus to cytoplasm, thus exerting its effect on inhibition of adiponectin secretion in adipocytes. Taken together, we demonstred that cytoplasmic RIP140 negatively modulates adiponectin secretion in adipocytes and provided evidence for targeting cytoplasmic RIP140 in adipocytes in promoting systemic insulin sensitivity.

NIH grants DK54733, DK60521, DA11190 and K02‐DA13926 to L.‐N.W.
Novel protein kinase D inhibitors cause potent arrest in cancer cell growth and motility
Authors: LaValle Courtney Rebecca , Bravo‐Altamirano Karla , Giridhar Karthik , Chen Jun , Sharlow Elizabeth R , Lazo John S , Wipf Peter , Wang Qiming Jane
Published: June 2021

Protein kinase D (PKD) has been implicated in a wide range of cellular processes and pathological conditions including cancer. However, targeting PKD therapeutically and dissecting PKD‐mediated cellular responses remain difficult due to lack of a potent and selective inhibitor. Previously, we identified a novel pan‐PKD inhibitor, CID755673, with upper nanomolar potency and high selectivity for PKD. To further enhance its selectivity and potency for potential in vivo use, five small molecule analogs with equal or greater potencies as CID755673 were generated and analyzed. Modifications to the aromatic core structure significantly increased potency while retaining high specificity for PKD. When tested in prostate cancer cells, all compounds inhibited activation of PKD1, with the compound kb‐NB142‐70 being most active. Importantly, the analogs caused a dramatic arrest in cancer cell proliferation and reduced viability. Cell migration and invasion were also inhibited by these analogs with varying potencies that correlated to their cellular activity. Throughout the range of experiments, the compounds kb‐NB142‐70 and kb‐NB165‐09 emerged as the most potent and specific analogs. In summary, our study reports a class of novel PKD small molecule inhibitors that are promising pharmacological tools for dissecting PKD function and for antagonizing aberrant PKD activity in cancer.
Targeting phosphodiesterase 7B and exchange protein directly activated by cAMP‐1 in chronic lymphocytic leukemia
Authors: Murray Fiona , Apaydin Eric , Zhang Lingzhi , Zahno Anja , Rassenti Laura , Kipps Thomas J , Insel Paul A
Published: June 2021

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B‐cells. We have shown that CLL is associated with increased mRNA expression of phosphodiesterase 7B (PDE7B), which catalyzes the hydrolysis of cAMP, and that PDE7 inhibitors promote apoptosis of CLL cells in a cAMP/protein kinase A‐dependent manner. In addition, we find that the expression of Exchange protein directly activated by cAMP (Epac), a downstream mediator of cAMP, is higher in CLL cells than in normal‐PBMC and B‐cells; its activation protects CLL cells from apoptosis. We investigated if the increase in PDE7B mRNA in CLL correlates with that of Epac‐1 and if targeted simultaneously would increase cAMP‐promoted apoptosis of CLL cells. We find that patients with CLL show a significant correlation between their fold‐increases in PDE7B and Epac‐1 mRNA (P<0.001, r=0.5395, n = 45). Inhibiting Epac‐1 or Epac‐1 signaling via siRNA (48 hr) or Rap‐1 inhibition (GGT1‐298, 5 μM, 48 hr), respectively, induced apoptosis in CLL cells and enhanced the pro‐apoptotic effect of PDE4/7 inhibition (IR‐284, 48 hr, 10nM). These data suggest that studies to define the regulation of PDE7B and Epac‐1 may provide insights into the pathophysiology of CLL and that decreasing the expression and function of Epac‐1 may be beneficial for treatment of CLL by increasing the pro‐apoptotic effects of PDE7 inhibitors. Supported by the Lymphoma and Leukemia Society and NIH.
The molecular mechanism of xanthohumol‐induced apoptosis in MCF‐7 and MCF‐7/ADR cells
Authors: Kang Youra , Park Min‐A , Kang Keon‐Wook , Kim Jung‐Ae
Published: June 2021

Xanthohumol, the principal prenylated chalcone that only found in the hop plant, Humulus lupulus L., has been shown to possess a variety of pharmacological activities. In the present study, we examined the xanthohumol induced apoptotic effects in breast cancer cells using wild‐type breast cancer cell (MCF‐7) and adriamycin resistant breast cancer cells (MCF‐7/ADR). Xanthohumol inhibited cell viability and induced apoptosis of both MCF‐7 and MCF‐7/ADR cells in a dose dependent manner. Interestingly, MCF‐7/ADR cells were more susceptible against xanthohumol than the wild‐type MCF‐7 cells as determined by MTT assay and PI staining. We found increased phosphorylation of STAT‐3 in MCF‐7/ADR cells which on xanthohumol treatment completely inhibited. Furthermore, MCF‐7/ADR cells were more sensitive to AG 1024, a selective inhibitor of IGF‐IR. The effect of xanthohumol on STAT‐3 inhibition was similar to that of AG 1024. In the down‐stream of STAT‐3, xanthohumol increased proapoptotic Bax and decreased antiapoptotic Bcl‐2 in MCF‐7, whereas xanthohumol decreased antiapoptotic Bcl‐x/L in MCF‐7/ADR. In case of caspase activity, xanthohumol induced cleavage of caspase‐9, ‐3 and ‐7 in MCF‐7/ADR cells and cleaved caspase‐9 in MCF‐7 cells. The apoptosis‐inducing activity of xanthohumol may be different depending on the cell type, especially in relation to STAT‐3 expression level.
NAD+ treatment can block rotenone‐induced nuclear apoptotic changes of PC12 cells
Authors: Hong Yunyi , Shao Jiaxiang , Xia Weiliang , Ying Weihai
Published: June 2021

Our previous study has shown that NAD+ can prevent astrocyte necrosis induced by oxidative stress and DNA alkylating agents. We have further found that intranasal administration of NAD+ can profoundly reduce ischemic brain damage. The goal of this current study is to determine the effects of NAD+ on the apoptotic changes of cells. We found that the mitochondrial Complex I inhibitor rotenone at 0.1 to 1 micromolar dose‐dependently induced nuclear condensation of PC12 cells: 0.75 micromolar rotenone induced an approximately 50% decrease in the nuclear size of the cells. Treatment of the cells with 0.25 to 1 mM NAD+ dose‐dependently attenuate rotenone‐induced nuclear condensation ‐‐‐ a hallmark of apoptosis: the rotenone‐induced nuclear condensation was nearly completely prevented by 1 mM NAD+. Our preliminary results have also shown that NAD+ treatment can prevent alkylating agent‐induced DNA laddering of astrocytes. Collectively, our results suggest that NAD+ treatment can significantly decrease toxic agent‐induced necrosis as well as apoptotic changes, thus providing information for understanding the mechanisms underlying the NAD+‐produced reductions of ischemic brain injury (Supported by a Key Research Grant of Shanghai Municipal Scientific Committee 08JC1415400 (WY), Pujiang Scholar Program Grant (WY)).
The mechanisms by which fenretinide and all‐trans RA induces apoptosis and differentiation, respectively in human HCC cells
Authors: Bushue Nathan , Wan Yu‐Jui Yvonne
Published: June 2021

Fenretinide [N‐(4‐hydroxyphenyl) retinamide] is a synthetic analog of all‐trans retinoic acid (RA). We have previously shown that fenretinide, but not all‐trans RA, induced apoptosis in Huh7 cells, a human hepatocellular carcinoma (HCC) cell line. While previous data shows that both differentiation and apoptosis are mediated through a retinoic acid receptor β (RARβ)‐dependent manner, the mechanisms by which fenretinide and all‐trans RA exert their different roles are not well understood. Our data showed that both chemicals induced RARβ mRNA and protein levels as well as activated ERK1/2 and Akt within 3 hrs. Both chemicals also induced the expression level of LC3. However, the conversion of LC3‐I to LC3‐II, a hallmark of autophagy, was substantially higher in all‐trans RA than fenretinide‐treated cells. In addition, the level of signaling adaptor p62, which is crucial for activation of death receptors and caspase 8‐mediated apoptosis, was significantly lower in all‐trans RA than fenrentinide‐treated Huh7 cells. Consequentially, fenretinide, but not all‐trans RA, caused ROS accumulation in Huh7 cells, which in turn lead to apoptosis. Taken together, the differential autophagy induction and p62 degradation might account for the different effect of all‐trans RA and fenretinide in controlling the survival or death of HCC cells.
Marine sponge Hymeniacidon sp. amphilectane metabolites potently inhibit rat brain microglia thromboxane B2 generation
Authors: Mayer Alejandro M , Chaudhry Saba , Hall Mary L , Aviles Edward , Rodriguez Abimael D
Published: June 2021

Neuroinflammation appears to involve release of thromboxane B2 (TXB2) and superoxide anion (O2−) by brain microglia (BMG). The purpose of this investigation was to determine the effect of five Hymeniacidon sp. amphilectane metabolites (HsH2.6, HsH1.11, HsH2.13, HsH2.29_456, and HsH2.11.X7.3) and two semi‐synthetic analogs (HsH1.11x and HsH1.11X2) on TXB2 and O2− generation from E. coli LPS‐activated rat BMG. Short and long term cell viability was assessed by lactate dehydrogenase (LDH) release (1.5 h) and mitochondrial dehydrogenase (MTH) activity (2.5–18 h), respectively. O2− levels were determined via superoxide dismutase‐inhibitable reduction of ferricytochrome C and TXB2 by EIA. Results were the following (n=3–4): all Hymeniacidon sp. metabolites and analogs potently inhibited TXB2 (IC50=0.20–5.69 μM) with low LDH release and minimal MTH inhibition. Comparison of IC50 of closely related amphilectane diterpenes HsH2.13 (IC50~0.20 μM) and HsH1.11 (IC50~0.23 μM) supports the observation that bioactivity is associated with presence of two isonitrile groups. However, the amphilectane diterpenoid skeleton plays a significant role, as suggested by comparison between IC50 of these two compounds and HsH1.11X2 (IC50~3.14 μM), where original isonitriles have been replaced by formamide groups. Lack of O2− inhibition would appear to suggest that all Hymeniacidon sp. metabolites and derivatives inhibit TXB2 synthesis by a cyclooxygenase‐dependent mechanism. Supported by Midwestern University and the RISE and SCORE Programs, University of Puerto Rico at the Río Piedras Campus.
Phagocyte Immunomodulatory Activity of Oenothein B, A Macrocyclic Elligatannin Isolated from Epilobium angustifolium
Authors: Schepetkin Igor A , Kirpotina Liliya N , Jakiw Larissa , Blaskovich Christie L , Jutila Mark A , Quinn Mark T
Published: June 2021

Epilobium angustifolium has been traditionally used to treat of a number of diseases; however, not much is known regarding its effect on innate immune cells. We found that extracts of E. angustifolium activated functional responses in neutrophils and monocyte/macrophages. Activity‐guided fractionation, followed by mass spectroscopy and NMR analysis, resulted in the identification of oenothein B as the primary component responsible for phagocyte activation. Oenothein B, a dimeric hydrolysable tannin, dose‐dependently induced a number of phagocyte functions in vitro, including intracellular Ca2+ flux, production of reactive oxygen species (ROS), chemotaxis, nuclear factor (NF)‐κB activation, and proinflammatory cytokine production. Furthermore, oenothein B was active in vivo, inducing keratinocyte chemoattractant (KC) production and neutrophil recruitment to the peritoneum after intraperitoneal administration. The ability of oenothein B to modulate phagocyte functions in vitro and in vivo suggests that this compound is responsible for at least part of the therapeutic properties of E. angustifolium extracts. This work was supported in part by NIH grants RR‐020185 and RR‐016455 and NIH contract HHSN266200400009C.
Mitochondrion is a novel site of biotransformation of dichloroacetate by glutathione transferase zeta
Authors: Li Wenjun , James Margaret O. , McKenzie Sarah , Stacpoole Peter W.
Published: June 2021

Dichloroacetate (DCA) is an environmental contaminant as well as a potential therapeutic agent through its ability to stimulate the pyruvate dehydrogenase complex. Repeated doses of DCA result in reduced drug clearance, probably through inhibition of glutathione transferase zeta 1 (GSTz1), a cytosolic enzyme that converts DCA to glyoxylate. Because DCA is known to be taken up into mitochondria, its principal site of action, it is of interest to explore the role of mitochondria in DCA biotransformation. Incubation of human and rat liver mitochondria with 14C‐DCA resulted in biotransformation only when glutathione was added, and glyoxylate was identified as the major metabolite. Protein expression of GSTz was detected in human and rat liver mitochondria by Western Blotting against human GSTz1 antibody. In rats, the expression of GSTz per gram of liver is 14 times as much in cytosol as in mitochondria. Similar to cytosolic GSTz, the expression and activity of mitochondrial GSTz was markedly reduced in rats treated with 500 mg DCA/kg for 8 weeks. Studies of sub‐mitochondrial fractions of rat liver showed that GSTz is localized in the mitochondrial matrix. We conclude that the mitochondrion is a site of DCA biotransformation catalyzed by GSTz, an enzyme shown for the first time to be co‐localized in cytosol and mitochondrial matrix. Supported in part by the US Public Health Service, ES 07355, ES 014617, and 1UL1 RR029890.
Residence of p450 system enzymes in endoplasmic reticular lipid microdomains
Authors: Brignac‐Huber Lauren , Reed Rob , Backes Wayne
Published: June 2021

P450 is part of an electron transport chain found in the endoplasmic reticulum (ER), with its catalytic function requiring interactions with other ER‐resident enzymes such as NADPH‐cytochrome P450 reductase (CPR). The goal of this study was to examine how the P450 system proteins are organized, and to determine if they are heterogeneously distributed in lipid microdomains (LM). ER from untreated and phenobarbital (PB)‐treated rabbits were partially solubilized with 1% Brij 98 for 5 minutes at 37°C, and LMs were isolated via sucrose gradient centrifugation. Lipid analysis showed that LM fractions were enriched in cholesterol and sphingomyelin, components that are also enriched in LMs found in plasma membrane and are indicative of lipid raft formation. Immune blotting of LM fractions showed that 75% of CYP1A2 and 40% of CPR resided in LM fractions in control ER. In PB microsomes, approximately 94%, 60%, and 65% of CYP1A2, CYP2B4, and CPR, respectively, were found in these regions. Preliminary studies showed that the lipid composition found in LM fractions has a significant effect on substrate metabolism, where the rate of CYP1A2 ethoxyresorufin metabolism was higher in vesicles with the same composition as LM, when compared to simple phosphatidylcholine vesicles. These results suggest that ER‐resident proteins exist in specific lipid microdomains that can influence P450 function. (Supported by NIEHS ES004344)
HPLC‐UV analysis of bupropion and hydroxybupropion: Application to in vivo pharmacokinetic drug‐drug interaction studies between bupropion and potent CYP2B6 inhibitors
Authors: Myers Alan L. , Molnari Jillissa C. , Moeller Bryant M. , Myers Alan L.
Published: June 2021

Bupropion is a popular antidepressant that is also prescribed in the management of smoking cessation. In humans, bupropion is predominantly metabolized to its active metabolite hydroxybupropion by CYP2B6. Inhibitors of CYP2B6 have the potential to decrease the clearance of bupropion, leading to adverse drug toxicity. We sought to develop a sensitive HPLC‐UV assay to quantify plasma and brain concentrations of bupropion and hydroxybupropion; and apply the assay to assess in vivo pharmacokinetic (PK) drug‐drug interaction (DDI) studies between bupropion and potent CYP2B6 inhibitors. Tissue extraction followed by HPLC‐UV detected timolol (IS), hydroxybupropion and bupropion at 6, 11 and 36 minutes, respectively. The LOD for both compounds was 6.0 ng/ml, and the intra‐day and inter‐day coefficients of variation was ±12% in plasma and ±15% in whole brain tissue. We then utilized this novel technique to evaluate the PK of bupropion and hydroxybupropion following repeated administration of the known CYP2B6 inhibitor ticlopidine (5 mg/kg daily x 5 days) in CF‐1 mice. Ticlopidine increased the plasma area under the concentration curve (AUC) of bupropion (2.0‐fold, p< 0.01) and decreased the plasma AUC of hydroxybupropion (1.2‐fold;p< 0.05). In whole brain tissue, ticlopidine increased the AUC of bupropion (1.3‐fold;p> 0.05)and decreased the AUC of hydroxybupropion (2.0‐fold;p< 0.001). In summary, we have developed a sensitive HPLC assay and suitable rodent model to evaluate in vivo PK DDI between bupropion and CYP2B6 inhibitors. Support: Drake Univ COPHS Intramural Research Grants.
Differential effects of Angiotensin II on intra‐renal hemodynamics in rats
Authors: Igbe Ighodaro , Oyekan Adebayo
Published: June 2021

Renal medullary blood flow (MBF) has been shown to differ qualitatively from cortical flow in response to certain vasoactive agents. The effect of AII infusion on intrarenal hemodynamics was investigated in rats testing the hypothesis that AT2 receptor mediates AII‐induced increase in renal MBF. AII (100, 300 and 1000 ng/kg/min) increased mean arterial blood pressure (MAP; 24±7%), decreased cortical blood flow (CBF; 30 ± 2%), but increased MBF (21±8%). Indomethacin (5 mg/kg), a COX inhibitor enhanced AII effects on MAP (154 ± 26%; p<0.05), MBF (141±46 %; p<0.05) and CBF (74±54%; p<0.05). NG nitro‐L‐arginine (L‐NNA), an inhibitor of NO synthase (100 mg/L orally) or 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ), a guanylyl cyclase inhibitor (2 mg/kg) enhanced AII effects on MAP (169±75 or 118±32%, p<0.05), CBF(107±50 or 85±47%, p<0.05) but blunted the effects of AII on MBF(150±21 or 96±15%, p<0.05). However, glibenclamide (20 μg/kg), a KATP+ channel blocker, was without effect. PD123319 (50 μg/kg/min), an AT2 blocker did not change basal or AII‐induced changes in MAP or CBF but blunted AII effects on MBF (60 ±11 %; p<0.05). CGP42112 (10 μg/kg/min), an AT2 agonist, reduced MAP but increased CBF and MBF ‐ effects were antagonised by PD123319. These findings demonstrate that AT2 receptors mediate AII‐induced increase in MBF by mechanisms involving guanylyl cyclase and NO but not prostanoids or KATP+ channels.
Synthesis, characterization and evaluation of derivates of 5‐aminosalicylic acid with antioxidant activity in inflammatory bowel disease.
Authors: Duran Leticia Araceli Ramirez , Ferrara José Guadalupe Trujillo , Rosales‐Hernández Martha Cecilia , Rosales Daniel Ramírez , Basurto José Correa
Published: June 2021

Recent theories suggest that the production of free radicals (RL) in IBD are directly related to the enzyme myeloperoxidase (MPO) and that this contributes to the damage The treatment of IBD is sulfasalazine, this is a conjugate of 5‐aminosalicylic acid, sulfapyridine linked to 5‐ASA acts as an antiinflammatory, MPO inhibitor as free radical stabilizer The aim of this work is to chemically synthesize amides and imides derived 5‐ASA evaluate whether extending the aromatic ring plays an important role in stabilizing RL, theoretically evaluate the interaction of the compounds on the MPO and select the most similar, and finally experimentally evaluate their activity as stabilizers Paramagnetic Resonance RL Electronics (RPE) The results showed that theoretically the related compounds on the MPO were those derived from maleic and succinic anhydrides. Subsequently synthesized and identified according to those reported in the literature and finally the results of EPR confirmed the antioxidant activity of 5‐ASA, however, to evaluate the synthesized compounds was observed that the imides showed a significant drop in the intensity of signals, which indicates that by extending the conjugation to the aromatic ring increases the ability to act as stabilizers RL.
Administration of ampicillin increases ileal Asbt protein amounts resulting in reduction of fecal bile acid excretion in mice
Authors: Yamazoe Yasushi , Takamatsu Yuki , Hamatsu Mayumi , Miyata Masaaki
Published: June 2021

Administration of the antibacterial drug ampicillin (ABPC) significantly decreased fecal bile acid excretion although it increased hepatic cholesterol 7α‐hydroxylation as well as CYP7A1 mRNA level. In the present study, we have investigated the mechanisms for the reduction of fecal bile acid excretion in ABPC‐treated mice. Bile acid amounts in small intestinal lumen and portal blood concentrations of bile acids were increased 1.6‐fold and 2.0‐fold, respectively, in ABPC‐treated mice. Ileal apical sodium‐dependent bile salt transporter (Asbt) mRNA levels were significantly increased in ABPC‐treated mice whereas ileal bile acid binding protein and organic solute transporter alpha and beta mRNA levels were reduced. Ileal Asbt protein amounts in brush border membranes were increased 2.5‐fold in ABPC‐treated mice. These results suggest that enhancement of ileal Asbt‐mediated bile acid absorption contributes to the reduction of fecal bile acid excretion in ABPC‐treated mice.
Leucine contents of isonitrogenous protein sources predict changes in body composition and muscle mass in rats
Authors: Norton Layne Eiseman , Layman Donald K , Wilson Gabriel J , Moulton Chris , Rupassara S. Indu , Garlick Peter J
Published: June 2021

Increases in intracellular leucine (leu) concentrations stimulate mTOR phosphorylation regulation of skeletal muscle protein synthesis (MPS) and leu content of isonitrogenous complete meals predict MPS potential of individual proteins (J Nutr 139:1103–9, 2009). While the molecular mechanism is established, direct effect of leu on long‐term changes in muscle weight or body composition are unknown. This study evaluated 4 isonitrogenous, isoenergtic diets containing wheat (supplemented with lys), soy, egg, or whey with 6.8, 8.0, 8.8, and 10.9% leu respectively, to determine meal effects of leu on signaling and MPS and long‐term impact on body composition. Male rats (275g) were fed 3 meals/d for 76d consisting of 16/54/30% of energy from protein, carbohydrates and fats. Blood and tissue were taken before or 90min after the breakfast meal at wk 2 and 11. Measurements included muscle weights, body composition by DXA, MPS by D5‐Phe incorporation, and phosphorylation of initiation factors 4E‐BP1 and S6K. Leu content of the meals increased muscle weights and was inversely correlated with bodyfat and these outcomes were associated with greater phosphorylation of 4E‐BP1 and S6K and MPS in animals fed egg and whey. These findings establish that leu content is a crucial factor defining the quality of proteins influencing muscle mass and body composition in adults. Supported by National Dairy Council and Egg Nutrition Center.

Grant Funding Source: National Dairy and Egg Nutrition Center
Liposomal Amikacin (Arikace™) in the Treatment of Lower Respiratory Tract Escherichia Coli in A Patient with Cystic Fibrosis
Authors: Laskoski John , Landon Chris
Published: June 2021

Objective

Aerosolized antibiotics are directed against Pseudomonas aeruginosa in cystic fibrosis (CF). E. coli decolonization was attempted in a colonized CF patient with multiple IV and oral drugs, including aminoglycosides, cephalosporins, and carbapenems prior to the use of Arikace™. We hypothesized that novel drug formulation nebulized liposomal Amikacin (Arikace™) will eliminate E‐coli from the lower airways.

Methods

Four Arikace™ treatment cycles were administered over a 7 month period in three treatment cycles of 28 d, and the fourth of 56 d. A non‐treatment cycle of 28 d was observed between each of the treatment cycles.

Results

Quantitative analysis of sputum confirmed a reduction in the bacterial load of one strand of E.coli from 2.4 x 10E6 cfu/gram to 1.0–10E3 cfu/gram. The density of a second strand of E. coli increased from16 x 10E6 cfu/gram to 18 x 10E6 cfu/gram post‐treatment. Decreased levels of multiple other pathogens were observed. The patient's sputum mass decreased from a pre‐treatment value of 11.1 g to 4.66 g following treatment.

Conclusions

Arikace™ is a valuable tool to decrease E. coli infection of the lower airways.
Separation, characterization, and proteomic analysis of hog‐barn dust fractions to identify lung disease‐relevant mediators
Authors: Dodmane Puttappa R. , Ciborowski Pawel S. , Schulte Nancy A. , Heires Art J. , Romberger Debra J. , Toews Myron L.
Published: June 2021

Workers in swine confinement facilities develop chronic inflammatory lung disease, and an aqueous hog‐barn dust extract (HDE) mimics disease‐relevant effects in cultured cells and animal models. We hypothesized that multiple components in HDE contribute to its effects on airway cells. The sensitivity/resistance of HDE activities for IL6/8 release and EGF receptor (EGFR) modulation in BEAS‐2B airway epithelial cells to chemical and enzyme treatments suggested heat‐stable proteins as mediators. Gel filtration revealed three separate peaks of activity for IL6/8 release, one >100 kDa, one ~30–50 kDa, and one of very low MW. Fractions of ~3–10 kDa had activity to phosphorylate and decrease binding to EGFRs. Mass spectrometry analysis of proteins in active fractions identified porcine pancreatic elastase (PPE). PPE mimicked some but not all cellular effects of complete HDE. Zymography identified protease activity in HDE, and protease inhibitors markedly reduced HDE activity on cells. Hog manure extract also exhibited activity on IL6/8 release and EGFRs. Thus multiple factors contribute to HDE effects, and manure‐derived PPE and/or other proteases are candidate mediators of hog‐barn dust‐induced lung disease. Supported by NIH R01‐OH008539.
Lymph Flow In Instrumented Dogs Varies With Exercise Intensity
Authors: Desai Pratikkumar Vrajeshbhai , Williams Arthur , Prajapati Parna R , Downey H. Fred
Published: June 2021

Objective

To determine the relationship between exercise intensity and thoracic duct lymph flow.

Methods

Five dogs were surgically instrumented with an ultrasonic flow transducer on the thoracic lymph duct and a catheter in the descending thoracic aorta. After recovery from surgery, data were collected at rest and as the dogs ran on a treadmill at speeds from 0 to 10 mph for 1 min at each speed with 15 min rest intervals.

Results

Heart rate increased significantly during exercise, whereas mean aortic pressure did not change. Resting lymph flow was 1.7 ± 0.2 ml/min. Exercise at 1.5 mph significantly raised lymph flow to 3.9 ± 0.6 ml/min (P < 0.01), 121% higher than resting flow. Lymph flow was further elevated at higher treadmill speeds, reaching 9.0 ± 1.6 ml/min (P < 0.01) at 10 mph, 419% higher than resting flow. Regression analysis demonstrated a linear relationship between treadmill speed and the percent increase in lymph flow (see Figure). After exercise, lymph flow returned rapidly to the resting value, but at higher speeds, lymph flow remained significantly greater than resting flow for 1 min (P < 0.01).

Conclusion

Exercise increases lymph flow, and this increase is positively correlated with exercise intensity. (Support: NIH grant U19 AT002023 and Osteopathic Research Center.)
Lymphatic Pump Treatment Increases Thoracic Duct Lymph Flow in Conscious Dogs with Edema Due to Constriction of the Inferior Vena Cava
Authors: Prajapati Parna Ramprasad , Shah Pankhil Virenkumar , King Hollis H. , Williams Arthur G. , Desai Pratikkumar Vrajeshbhai , Downey H. Fred
Published: June 2021

Objective

To examine the effects of osteopathic lymphatic pump treatment (LPT) on lymph flow in the thoracic duct of instrumented conscious dogs in the presence of edema produced by constriction of the inferior vena cava (IVC).

Methods

Six dogs were surgically instrumented with an ultrasonic flow transducer on the thoracic lymph duct and catheters in the descending thoracic aorta and in the IVC. After post‐operative recovery, lymph flow and hemodynamic variables were measured 1) pre‐LPT, 2) during 4 min LPT, 3) post‐LPT, in the absence and presence of edema produced by IVC constriction.

Results

IVC constriction increased abdominal girth from 60±2.6 to 75±2.9 cm. Before IVC constriction, LPT increased lymph flow (P<0.05) from 1.9±0.2 ml/min to a maximum of 4.7±1.2 ml/min, whereas after IVC constriction, LPT increased lymph flow (P<0.05) from 7.9±2.2 to a maximum of 11.7±2.2 ml/min. The incremental lymph flow mobilized by 4 min of LPT, i.e., the flow that exceeded 4 min of baseline flow, was 10.6 ml after IVC constriction (not different from before IVC constriction).

Conclusions

Edema caused by IVC constriction markedly increased lymph flow in the thoracic duct. LPT increased thoracic duct lymph flow before and after IVC constriction. The lymph flow mobilized by 4 min of LPT in presence of edema was not significantly greater than that mobilized prior to edema. (Support: NIH grant U19 AT002023 and Osteopathic Research Center.)
Elucidating the sources of enhanced oxygen consumption during early reperfusion following skeletal muscle ischemia in the rat spinotrapezius muscle
Authors: Nugent William , Golub Alexsander , Pittman Roland
Published: June 2021

Prolonged skeletal muscle ischemia is disruptive to cellular respiration. Upon reperfusion, the metabolic rate of the affected tissue is altered in a fashion dependent on the duration and totality of ischemia. This can be used as an indicator of the changes undergone in cellular metabolism. We have employed Phosphorescence Quenching Microscopy in conjunction with a flow‐arrest technique to assess the influences of external, pressure‐induced 1‐ to 30‐min focal ischemic durations on interstitial oxygenation (PO2) and the consumption of oxygen (VO2) in spinotrapezius muscles of SD rats. VO2 was assessed by the rate of PO2 decline during brief flow‐arrest compressions during reperfusion. Our tests of this intermittent compression technique have indicated that 5 s of flow‐arrest followed by 15 s of flow restoration allow for measurement of VO2 without compromising baseline or reperfusion recovery of interstitial PO2. Our preliminary studies show a ~5‐fold increase in VO2 following 30 min of ischemia, which then follows an exponential decay over several minutes back to baseline levels. Chemical modification of mitochondrial function, [NO], and [ROS] are expected to reveal the sources of this VO2 spike and how they influence the return to baseline. As hypoxia is central to I/R injury, a description of mitochondrial function during this insult will be helpful.
Exercise training improves oxygen consumption and exercise performance in Obese Zucker rats
Authors: Jones Louis , Fuller William , Xiang Lusha , Hester Robert
Published: June 2021

Exercise is an important therapy for decreasing body weight and improving health. We hypothesized that exercise training would improve oxygen consumption and exercise capacity in obese Zucker rats. Six week old lean and obese rats were divided into exercise and control groups (n=5 for each group). Rats were trained on treadmill for 30 minutes per day, 4 days/week for 4 weeks. On the 5th day of each week oxygen consumption (VO2, mL/Kg/min) and endurance were measured starting at a speed of 14m/min and increasing 2 meters every 2 minutes until the rats were exhausted. There were no differences in weight between the sedentary and exercise leans or between the obese groups. VO2 in the lean exercise group at week 1 was 40±1 at 14 m/min and 58±1 at 32 m/min. At week 4 VO2 in the lean exercise group was 41±3 at 14 m/min and 60±6 at 32 m/min. VO2 in the obese exercise group at week 1 was 41±1 at 14 m/min and 50±2 at 20 m/min. At week 4 VO2 in the obese exercise group was 37±1 at 14 m/min and 54±6 at 20 m/min. VO2 in a sedentary 10 week old lean group was 52±3 at 14 m/min and 72±1 at 30 m/min. VO2 in a sedentary 10 week old control obese group was 39±2 at a maximum running speed of 14. Thus exercise training improves the aerobic capacity of obese Zucker rats, allowing them to work at a higher rate than their untrained counterparts.
Determinants of Leukocyte Margination in Rectangular Microchannels
Authors: Munn Lance L , Jain Abhishek
Published: June 2021

Polydimethylsiloxane (PDMS) devices have provided a new set of tools for studying fluid dynamics of blood at the scale of real microvessels. However, we are only starting to understand the power and limitations of this technology. To determine the applicability of PDMS microchannels for blood flow analysis, we studied white blood cell (WBC) margination in channels of various geometries and blood compositions. We found that WBCs prefer to marginate downstream of sudden expansions, and that red blood cell (RBC) aggregation facilitates the process. In contrast to tubes, WBC margination was restricted to the sidewalls in our low aspect ratio, pseudo‐2D rectangular channels and consequently, margination efficiencies of more than 95% were achieved in a variety of channel geometries. In these pseudo‐2D channels blood rheology and cell integrity were preserved over a range of flow rates, with the upper range limited by the shear in the vertical direction. We conclude that, with certain limitations, rectangular PDMS microfluidic channels are useful tools for quantitative studies of blood rheology. Funded by NIH R21CA126761.
Shear stress Lp response enhanced by chondroitin sulfate and hyaluronic acid is Rho kinase dependent
Authors: Adamson R H , Sarai R K , Altangerel A , Clark J F , Curry F E
Published: June 2021

Vascular endothelial cells (EC) orient their long axes parallel to the fluid flow direction both in vivo and in culture. The surface glycocalyx of cultured EC transduces the fluid flow shear stress that mediates nitric oxide production and EC cytoskeletal rearrangement (Thi et al., PNAS 2004 101(47): 16483). Previously we showed that for intact microvessels perfusion against the normal flow direction stimulated increased hydraulic permeability (Lp) correlated with shear stress and induced rearrangement of cytoskeletal structures using Ringer's solution containing bovine serum albumin (BSA; 10 mg/ml) as perfusate. As a further test we added chondroitin sulfate (C; 0.2 mg/ml) and hyaluronic acid (H; 0.2 mg/ml) to the perfusate (CH; to maintain glycocalyx structure; Potter & Damiano, Circ Res 2008; 102:770) and we measured Lp in rat mesentery venular microvessels perfused for 2 hours against the normal flow direction (Reverse). Reverse perfused CH vessels had a mean Lp change (ΔLp) of 18 ± 9 (sem; ×10−7 cm/(s×cmH2O)) while for Reverse perfused BSA vessels ΔLp was 7.1 ± 3.5. Forward perfusion did not change Lp in either group. For Reverse perfusion using CH and the Rho kinase inhibitor Y27632 (30 μM) ΔLp was 6.2 ± 3.5. The data suggest that CH helps maintain an intact glycocalyx which is more sensitive to altered shear stress than that of BSA perfused vessels and that the ΔLp response is Rho kinase dependent. Supp NIH HL44485.
Loss of glycocalyx barrier properties impairs insulin‐mediated recruitment of microvascular blood volume in rat hind limb muscle
Authors: Eskens Bart , Cobelens Hanneke , Vink Hans , VanTeeffelen Jurgen
Published: June 2021

Experimental data suggest that a rapid increase in capillary blood volume underlies the insulin‐mediated glucose uptake in skeletal muscle. We hypothesize that loss of glycocalyx properties impairs the ability of insulin to increase microvascular blood volume in skeletal muscle.

In anesthetized rats, the hind limb muscle microcirculation was visualized with a Sidestream Dark Field camera before and during a 30 minute infusion of insulin (6mU/min/kg). Median values and dynamic range of red blood cell (RBC) column widths were measured to determine functional microvascular diameters and glycocalyx barrier function, respectively.

In control rats (n=5), insulin infusion increased within five minutes the dynamic range of RBC width with 0.56 μm ± 0.19 μm (mean ± SEM) (P<0.05). Hereafter, microvascular diameter increased with 0.69 μm ± 0.22 μm (P<0.05), while the dynamic range of RBC width returned to pre‐insulin values. In rats, in which the glycocalyx was degraded with a bolus of hyaluronidase (500U; n=7), the dynamic range was already increased at baseline levels with 0.30 μm ± 0.02 μm (p<0.05), with no further effect of insulin. Rapid insulin‐mediated increases in microvascular blood filling in rat skeletal muscle appear to be preceeded by an attenuation of glycocalyx barrier properties. This ability of insulin is impaired after enzymatic degradation of the glycocalyx.

Supported by DFN 2006.00.027 and NHF 2005T037
Glycated proteins inhibit K channels in isolated vascular smooth muscle cells
Authors: Yang Yan , de Dios Stephanie T , Jenkins Alicia J , Davis Michael J , Hill Michael A
Published: June 2021

Fibronectin (FN) has been shown to enhance K+ channel activity via an integrin‐mediated mechanism. As vascular smooth muscle (VSM) K+ channels mediate vasodilation, we investigated whether advanced glycation of fibronectin (as occurs in diabetes and renal failure) alters the normal stimulatory effect of this matrix protein on these channels. Under sterile conditions, FN (1mg/ml) was glycated (gFN) for 5 days in the presence of methylglyoxal (50mM) or glycolaldehyde (50mM). Albumin, a non‐matrix protein, was similarly glycated as a control. VSM cells were enzymatically isolated from rat cerebral arteries for K+ channel activity studies via whole cell patch clamp. The inhibitors, iberiotoxin (0.1μM) and 4‐aminopyridine (1mM), were used to identify contributions of large conductance, Ca2+‐activated, K+ channels and voltage‐gated K+ channels, respectively. While native FN enhanced whole cell K+ current (1.8 fold), gFN caused a 56% inhibition of current compared to baseline. Furthermore, native albumin did not enhance basal K+ current but when glycated caused inhibition (61%; p < 0.05). Inhibitor studies indicated a predominant effect of gFN on the Kv component of total K+ current. These studies provide a potential mechanism by which advanced glycated proteins impair VSM function and adversely impact arteriolar vasodilation.