bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Arachidonic Acid Metabolism in the Rat Kidney by Thymoquinone: Implications for Regulation of Blood Pressure
Authors: Elbarbry Fawzy , Ung Aimy , Lack Kelsey
Published: June 2021

Background

We investigated the effects of thymoquinone (TQ), a Quinone phytochemical in the plant Nigella Sativa, on Arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as our animal model system.

Methods

Rats were treated for 7 weeks with TQ (10, 20, or 40 mg/kg) added to their drinking water; drinking water alone was used as our control. Mean arterial pressure (MAP) was measured at 7‐day intervals throughout the study. At the end of the treatment, rats were euthanized, followed by preparation of kidney microsomes to measure the activity (using LC/UV and fluorescence assay) and expression (using immunoblotting) of enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20‐hydroxyeicosatetraenoic acid, and the soluble epoxide hydrolase (sEH), which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids.

Results

We found that treatment with TQ leads to significant reductions in both the expression and activity of renal CYP4A isozyme. Only the high dose TQ resulted in a significant reduction in the expression and activity of renal sEH. Consistent with these data, we have found that treatment with TQ resisted the progressive rise in MAP in the developing SHR in a dosedependent manner.

Conclusion

This is the first demonstration that TQ modulates the metabolism of AA by both kidney P450 enzymes and sEH in SHR rats. This may represent a novel mechanism by which TQ protects SHR rats against the progressive rise in blood pressure.

Support or Funding Information

Pacific University Research Incentive Grant

Medical Research Foundation of Oregon

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective effect of APLN against liver X receptor‐mediated hepatic steatosis through HG11/APLNR in human and mouse hepatocytes
Authors: Huang Jin , Kang Saeromi , Park Soo‐Jin , Im Dong‐Soon
Published: June 2021

Background

Non‐alcoholic fatty liver disease is the most commonly occurring chronic liver disease, and hepatic steatosis is associated with obesity and type II diabetes. APLN is an adipokine that acts on a G protein‐coupled receptor named HG11/APLNR, and has been established to play pivotal roles in various physiological conditions. However, the function of APLN in hepatocytes has not been fully investigated.

METHODS

In order to assess the functional roles of APLN and APLNR in hepatocytes, we used an in vitro model of liver X receptor (LXR)‐mediated hepatocellular steatosis.

RESULTS

In Hep3B human hepatoma cells, T0901317 (a specific LXR activator) induced lipid accumulation and this was inhibited by APLN. T0901317 also induced the expression of SREBP‐1c, a key transcription factor for lipogenesis. APLN not only inhibited SREBP‐1c induction at the mRNA and protein levels but also induced lipolytic PPAR alfa expression. Furthermore, these protective effects of APLN were inhibited by APLN‐F13A (a specific APLNR antagonist). Furthermore, silencing of APLNR by siRNA transfection also inhibited the actions of APLN. Specific inhibitors of cellular signaling components showed inhibition of lipid accumulation by APLN was mediated through Gi/o proteins, AMPK, and SREBP‐1c suppression during the early stage and through AMPK, ERKs, and PPAR alfa induction during the late stage. In addition, the protective effect of APLN was confirmed in mouse primary hepatocytes.

CONCLUSION

The present findings suggest APLN‐APLNR signaling in hepatocytes functions to protect against lipid accumulation in liver through two signaling pathways, that is, via AMPK activation and PPAR alfa induction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of Pregnane X Receptor Sensitizes Mice to Hemorrhagic Shock Induced Liver Injury
Authors: Xie Yang , Xu Meishu , Deng Meihong , Li Zhigang , Wang Pengcheng , Guo Yan , Ma Xiaochao , Fan Jie , Billiar Timothy , Xie Wen
Published: June 2021

Hemorrhagic shock (HS) is a life‐threatening condition often associated with traumatic injuries. Hemorrhagic shock causes tissue hypoperfusion, which may lead to injury to multiple organs including the liver. Pregnane X receptor (PXR) is a xenobiotic receptor that regulates the expression of drug‐metabolizing enzymes (DMEs) such as CYP3A. Many of the drugs used in the trauma patients are known to activate PXR and induce CYP3A. However, it is unclear whether and how PXR plays a role in the regulation of DMEs in the setting of HS and if so, whether activation of PXR beneficial or detrimental to the HS‐responsive liver injury. In this study, we showed that genetic or pharmacological activation of PXR sensitized mice to HS‐responsive liver injury, whereas ablation of PXR protected liver from damage following HS. Mechanistically, the sensitizing effect of PXR activation may have been accounted for by the PXR‐responsive induction of Cyp3a and increased oxidative stress in the liver. Pharmacological inhibition of Cyp3a attenuated the sensitizing effect of PXR. Treatment with the antioxidant NACA relieved the oxidative stress and protected mice from HS‐responsive liver injury. Our results suggest that the unavoidable use of PXR‐activating drugs in trauma patients has the potential to exacerbate HS‐responsive liver injury, which can be mitigated by the co‐administration of anti‐oxidative agents.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Carbazeran as an Enzyme‐Selective Substrate of Human Aldehyde Oxidase: Evidence for Regiospecific Oxidation of Carbazeran by CYP1A2, CYP2D6, CYP3A4, and CYP3A5
Authors: Lau Aik Jiang , Xie Jiarong , Saburulla Nur Fazilah D/O , Chen Shiyan , Zhang Harris L. , Wong Siew Ying
Published: June 2021

Aldehyde oxidase‐1 (AOX‐1) is a cytosolic xenobiotic‐metabolizing enzyme that has gained increasing interest because of its emerging role in drug metabolism and toxicity. It is a catalyst of carbazeran 4‐oxidation, resulting in the formation of 4‐oxo‐carbazeran. However, cytochrome P450 is also a catalyst of oxidative reactions. Therefore, we tested the hypothesis that cytochrome P450 and AOX‐1 catalyze carbazeran 4‐oxidation in the endoplasmic reticulum and cytosol, respectively. Incubations containing human liver microsomes with or without NADPH yielded 4‐oxo‐carbazeran, but to a lesser extent than that in incubations containing human liver cytosol. Cytochrome P450 inhibitors (1‐aminobenzotriazole and ketoconazole) did not decrease the extent of carbazeran 4‐oxidation in liver microsomes, whereas an AOX‐1 inhibitor (hydralazine) decreased it in liver cytosol. Human recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 did not catalyze carbazeran 4‐oxidation, in contrast to the catalysis by recombinant AOX‐1. The extent of carbazeran 4‐oxidation in incubations containing liver microsomes was attributed to the presence of AOX‐1 in the microsomal preparations, as indicated by the decrease in carbazeran 4‐oxidation by an AOX‐1 inhibitor (hydralazine) and the detection of AOX‐1 protein in liver microsomes (at a level that was up to one‐third of that in liver cytosol). Cytosolic sulfotransferases‐catalyzed dehydroepiandrosterone sulfation increased with increasing amount of microsomes, indicating that the two lots of hepatic microsomes are contaminated with not only AOX‐1, but also other cytosolic enzymes. An isomer of 4‐oxo‐carbazeran, putatively identified as 5‐hydroxy‐carbazeran or 8‐hydroxy‐carbazeran, was detected in incubations containing carbazeran and CYP1A2, CYP2D6, CYP3A4, or CYP3A5. The 5‐hydroxy‐carbazeran or 8‐hydroxy‐carbazeran peak was detected in human liver microsomal incubation and the peak area increased with increasing incubation time. The 5‐hydroxy‐carbazeran or 8‐hydroxy‐carbazeran peak was decreased by a pan‐CYP inhibitor (1‐aminobenzotriazole), but not by an AOX‐1 inhibitor (hydralazine). The addition of 1‐aminobenzotriazole increased the 4‐oxo‐carbazeran formation, indicating a shunting of carbazeran metabolism to AOX‐1 in the presence of the CYP inhibitor. In conclusion, carbazeran is a dual substrate of AOX‐1 and cytochrome P450. However, AOX‐1, but not cytochrome P450, catalyzes carbazeran 4‐oxidation.

Support or Funding Information

Singapore Ministry of Education Academic Research Fund Tier 1 [Grant R‐148‐000‐218‐112 to AJL]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physical Organization of Heme Oxygenase 1, NADPH‐Cytochrome P450 Reductase, and the Cytochromes P450 in the Endoplasmic Reticulum
Authors: Connick John Patrick , Backes Wayne L.
Published: June 2021

NADPH‐Cytochrome P450 reductase (POR) is an endoplasmic reticulum(ER)‐bound enzyme responsible for providing electrons to reactions catalyzed by the cytochromes P450 (P450s) as well as heme oxygenase‐1 (HO‐1). Previous work has revealed that some P450s form physical complexes with each other, leading to changes in their interactions with POR and subsequent modification of P450 activities. Because HO‐1 is expressed in the same location and must compete with the P450s for POR binding, we hypothesized that increased HO‐1 expression could inhibit the POR‐binding of individual P450s and that increased P450 expression could inhibit the POR‐binding of HO‐1. Additionally, HO‐1 has the potential to form homomeric complexes or heteromeric complexes with individual P450s as we have seen previously with the P450s. The goal of this study was to better understand the organization of these proteins, how they might interact in the membrane environment, and to determine if these interactions affected protein function. Bioluminescence resonance energy transfer (BRET) was used to examine these potential protein‐protein interactions. BRET involves expressing a pair of proteins, one with a green fluorescent protein tag and one with a Renilla luciferase tag, in HEK293T cells. Because energy transfer between the two protein tags requires them to be in close proximity, we can determine whether the tagged proteins exist as stable complexes in the cells. Co‐transfecting a third, untagged protein allows us to determine whether this protein destabilizes the complex formed by the tagged proteins. To date, we have determined that HO‐1 forms homomeric complexes that can be disrupted by excess POR, and that HO‐1 appears to form complexes with CYP1A1, CYP1A2, and CYP2D6, but not with CYP2A6 or CYP2C9. At least the HO‐1•CYP1A1 complex appears to be stable in the presence of POR. While further work is required to paint a complete picture of this system, the early results suggest the presence of a previously undescribed network of protein‐protein interactions involving HO‐1, POR, and the P450s.

Support or Funding Information

NIH NIEHS ES004344

NIH NIEHS ES013648

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of the enzyme isoforms responsible for the N‐oxide detoxification pathway in tobacco specific nitrosamines (TSNAs)
Authors: Perez‐Paramo Yadira Xitlalli , Watson Christy J. W. , Chen Gang , Lazarus Philip
Published: June 2021

Tobacco‐specific nitrosamines (TSNAs), are among the most potent carcinogens found in tobacco products. Among them are N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), N′‐nitrosoanabatine (NAT), 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and 4‐ (methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), a major metabolite of NNK. TSNAs are detoxified by a number of metabolic pathways, including glucuronidation by the UDP‐glucuronosyltransferase (UGT) family of enzymes, as well as N‐oxidation by an as yet unidentified enzyme or family of enzymes. TSNAs share a pyridine ring in their chemical structure that can be further metabolized to a detoxification N‐oxide product. TSNA‐N‐oxides have been identified in the urine of rodents exposed to TSNAs and NNAL‐N‐oxide was found in the urine of smokers, however, the enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors for the major hepatic cytochrome P450 (CYP) enzymes (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in oxidation reactions with pooled human liver microsomes, including phenacetin, coumarin, bupropion, amodiaquine, diclofenac, omeprazole, dextrometrphan, chlorzoxazone and midazolam. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors for CYPs 1A2, 2A6, 2E1, 2C19 and 3A4. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation was examined using microsomal fractions from CYP over‐expressing HEK293 cells. Microsomes from cells over‐expressing CYPs 1A2, 2C19, 2E1 and 3A4 isoforms exhibited TSNA‐N‐oxide formation. The isoforms 2C19 and 2E1 presented an approximate two‐fold higher activity than the other active isoforms. These data suggest that several cytochrome P450 enzymes may play an important role in the N‐oxidation of TSNAs, and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk.

Support or Funding Information

National Institutes of Health, National Institutes of Environmental Health Sciences [Grant R01‐ES025460]

Health Sciences and Services Authority of Spokane, WA [Grant WSU002292]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioengineered let‐7c is Effective at Reducing Orthotopic Hepatocellular Carcinoma Tumor Burden and is Well Tolerated in Mouse Models
Authors: Jilek Joseph Lionel , Zhang Qianyu , Ho Pui Yan , Tu Meijuan , Duan Zhijian , Yu Aiming
Published: June 2021

Given pleiotropic molecular targeting mechanisms, restoration or inhibition of endogenous microRNAs (miRNA) is a promising pharmacological strategy. Recently, our group has bioengineered a collection of miRNA agents in live cells using a non‐coding carrier RNA (nCAR) based technology. In vitro screening identified nCAR/let‐7c as the most potent at inhibiting hepatocellular carcinoma (HCC) viability; this was found to be processed to the mature let‐7c molecule and suppressed target gene expression. As such, in vivo efficacy and safety of this agent was characterized in the present study using polyethyleneimine (PEI) and liposomal‐polyplex (LPP) RNA formulations. Huh7 cells stably expressing luciferase and green fluorescent protein were orthotopically engrafted into immunocompromised mice. To evaluate efficacy, 40 μg of nCAR/let‐7c or a truncated control agent was administered systemically every‐other day for two weeks, followed immediately by necropsy. Live animal bioluminescent and ex vivo fluorescent imaging suggested that LPP‐formulated nCAR/let‐7c reduced tumor burden to a greater degree than all controls and PEI‐formulated nCAR/let‐7c. Additionally, both nCAR/let‐7c and truncated control agents were well‐tolerated compared to untreated mice, as evidenced by gross evaluation and similar bodyweights throughout the study; however, further studies are warranted in immunocompetent animal models to elucidate the complete toxicological profile and immunogenic potential of nCAR/let‐7c. These data are the first to demonstrate that let‐7c restoration using a biologically‐derived agent can serve as a potent antineoplastic agent, presenting a new, clinically‐relevant strategy for HCC therapy.

Support or Funding Information

This work was supported by National Institutes of Health grants [R01GM113888 and U01CA175315 to A.‐M.Y.] and an institutional Pharmacology Training Program grant [T32GM099608 to J.L.J.].

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeted Osmotic Lysis of Highly Invasive Carcinomas Using a Pulsed Magnetic Field and Pharmacological Blockade of Voltage‐Gated Sodium Channels
Authors: Paul Dennis , Maggi Paul E. , Minkoff Lawrance , Piero Fabio Del , Gould Harry J.
Published: June 2021

Simultaneous electrical or physiological stimulation of voltage‐gated sodium channels (VGSCs) and concomitant pharmacological blockade of sodium, potassium‐ATPase (sodium pumps) causes lysis of cells that overexpress VGSCs. Lysis does not occur in tissues that express normal levels of VGSCs. Metastatic carcinomas overexpress VGSCs. Pulsed magnetic fields (PMFS) will also activate VGSCs. Accordingly, we hypothesized that a PMF would lyse invasive carcinomas concomitantly treated with the sodium pump blocker, digoxin, without affecting noncancerous cells or tissue. MDA‐MB‐231 breast cancer cells cultured in DMEM, or MCF‐10a normal breast epithelial cells cultured in MEGM, were dissociated with Cellstripper and resuspended in microfuge tubes with or without 5–500 nM digoxin. Microfuge tubes were placed in an 8″l × 2.5″dia. electromagnetic solenoid with 697 turns of 12 ga copper wire. A Tektronix AFG 3021B waveform generator provided a 2 Vpk – 5 Vpk, 25 Hz pulse. The signal was fed into an AE Techron 7224 DC amplifier connected between the solenoid and the function generator, producing 9 Apk – 22 Apk monophasic DC current pulses into the solenoid, resulting in a 36 to 90 mT PMF inside the solenoid. After stimulation, cells were fixed, stained with cresyl violet and percent nonviable cells counted. Digoxin treated MDA‐MB‐231 cells lysed dose‐ and stimulation‐dependently within the 10 min stimulation interval, whereas drug‐only and stimulation‐only controls were unaffected. Neither digoxin‐treated nor untreated MCF‐10a cells lysed. For in vivo experiments, J/Nu mice with MDA‐MB‐231 xenografts were injected with 5 mg/kg digoxin or saline 5 times at 1 hr intervals on days 1, 3 and 5. This protocol achieves steady‐state pharmacokinetics. On each of the stimulation days, mice were confined to the solenoid and exposed to the 25 Hz, 90 mT PMF for 15 min starting 30 min after the last injection. Mice were euthanized and fixed with paraformaldehyde 24 hrs after the last treatment. Subcutaneous tumors from mice treated with digoxin and PMF were characterized by 60–100% tumor lysis. No lysis was observed in any of the normal tissues of treated mice. Drug‐only and stimulation‐only controls did not differ from untreated controls. These results are evidence that targeted osmotic lysis using a PMF warrants further study as a treatment for metastatic carcinomas.

Support or Funding Information

Supported by the LSUHSC Depts. of Pharmacology and Neurology and a research contract with Oleander Medical Technologies.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Dose Ascorbate Effect on Certain Cancer Cell Lines
Authors: Bennett Lunawati Lo
Published: June 2021

In recent years, the use of natural dietary antioxidants in cancer treatment has gained popularity due to their minimal cytotoxic effects with no evidence of causing damage to normal cellular tissues. Since the discovery of vitamin C (ascorbic acid, ascorbate), this antioxidant has a controversial history in relation to cancer treatment. Oral ascorbate had no or little effect on survival of the cancer patients. However, the discovery that intravenous ascorbate can provide 100‐fold higher plasma levels as compared to oral intake has renewed interest on the role of ascorbate. One hypothesis to support the use of high‐dose ascorbate is the finding that at high dose (pharmacological concentrations); it may act as a prodrug to deliver extracellular H2O2 that is selectively toxic to cancer cells. High dose ascorbate is non‐toxic and well tolerated to normal cells.

In the present study, several cell lines such as HepG2 (human liver carcinoma cells), MCF7 (human breast cancer), SaOS2 (human osteosarcoma), and CaCO2 (human colorectal adenocarcinoma cells) were used. Ascorbate was buffered to pH 7 with sodium hydroxide and prepared immediately before use. Cells viability measured by MTT assay (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyl‐tetrazolium bromide), Hoechst33342, H2DCFDA, and Rhodamine123 staining using fluorescent microscope were conducted to determine cell viability, chromatin inducing apoptosis, intracellular generation of ROS, and mitochondrial membrane potential, respectively. Western Blot to detect different signaling protein expressions such as Poly (ADP‐Ribose) polymerase (PARP), Casp‐3, Casp‐9, P‐53, Bax, Bcl2, Apaf‐1, MMP2, MMP 9, BRCA1, BRCA2, Bim, P‐Map kinases were conducted to test the hypothesis that ascorbate decreased cell proliferation, increased intracellular ROS generation and apoptotic cells, and decreased mitochondrial membrane potential. The concentration of ascorbate that causing 70–80% death of the cells ranged from 0.8 mM in SaOS2, 1.5 mM in CaCO2, 6 mM in HepG2, and 7 mM in MCF7, respectively. This finding suggests that high dose ascorbate has a potential candidate as effective anti‐neoplastic in several cancer types.

Support or Funding Information

Union University College of Pharmacy

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomarker Discovery for Cancer Sensitivity to CDK8 Inhibitors
Authors: Lu Zhixin , Spear Jensen E. , Russu Wade A.
Published: June 2021

Background

A novel piperazinylpyrimidine compound, Q12, was synthesized and tested for its potential to selectively inhibit the growth of certain tumor cell lines within the NCI‐60 cell line panel. Q12 is an ATP competitive inhibitor of cyclin dependent kinase 8 (CDK 8). We studied Q12 in the context of sensitive cell lines MDA‐MB‐468 (GI50 0.06 μM) and HCT‐116 (GI50 0.27 μM) and a non‐sensitive cell line COLO 205 (GI50 30 μM). The MDA‐MD‐468 cell line represents triple negative breast cancer, a difficult type of cancer to treat because of lacking HER2, estrogen and progesterone receptors. HCT‐116 and COLO 205 are human colon cancer cell lines, which harbor β‐catenin and APC mutations respectively. We hypothesized that Q12 is a promising molecule that can be used to probe biomarker discovery for CDK8 inhibitor sensitive tumor types.

Material and Methods

In vitro studies were performed by using of MDA‐MB‐468 breast cancer cell line, HCT‐116 and COLO 205 colon cancer cell lines. GI50 was measured and apoptosis assay was performed. The growth inhibitory effect of Q12 was assessed by Muse Cell Viability Kit. To evaluate the effects of Q12 on β‐catenin and STATs, Western Blotting were used.

Results

The cell viability essay shows Q12 potently inhibited the growth and viability of MDA‐MB‐468 cells and HCT‐116 cells, but COLO 205 cell line was resistant. Western blotting shows the reduction of STAT1 phosphorylation, a robust indicator of CDK8 target engagement, in all three cell lines upon Q12 treatment. Western blotting also showed a reduction in β‐catenin in both HCT‐116 and COLO 205 cells, but unchanged in MDA‐MB‐468 cell line, upon treatment with Q12.

Conclusion

Our data shows that active β‐catenin is not necessarily a driver of colon tumor cell proliferation and viability nor is it a marker for CDK8 inhibitor sensitivity. Both HCT‐116 and COLO205 cells harbor mutations that stabilize β‐catenin (β‐catenin and APC respectively), and both experience reductions in β‐catenin upon treatment with CDK8, but have vastly different proliferation and viability responses. The MDA‐MD‐468 cell line is very sensitive to Q12, but does not experience a reduction in β‐catenin. What is interesting to note is that both HCT‐116 and MDA‐MB‐468 harbor mutations related to RB1 (CDKN2A and RB1 respectively). Disparate E2F1 transcriptional activity may hold the key to discovery of a biomarker of CDK8 inhibitor sensitivity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysosomal Ca2+ Release via TRPML1 Channels Regulated by Acid Ceramidase and Associated Sphingolipids in Podocytes
Authors: Li Guangbi , Li Ningjun , Li Pin‐Lan
Published: June 2021

Transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca2+ release that is involved in Ca2+‐dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested the hypothesis that acid ceramidase (AC) plays a pivotal role in regulation of TRPML1 channel activity in podocytes. To detect TRPML1 channel‐mediated Ca2+ release, cultured murine podocytes were transfected via nucleofection with GCaMP3‐ML1, a TRPML1‐targeted genetically encoded Ca2+ indicator. To characterize GCaMP3 expression in lysosomes, confocal microscopy confirmed the colocalization of GCaMP3‐ML1 and Lamp‐1 suggesting its successful transfection and localization in lysosomes. ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased GCaMP3 fluorescence intensity in podocytes. For dynamic recording of Ca2+ release, a high speed wavelength switching fluorescent microscopic imaging system was used to continuously monitor GCaMP3 fluorescence signal in living podocytes. It was found that in the presence of a lysosome‐disrupting cathepsin C substrate, GPN that depleted lysosomal Ca2+, GCaMP3‐detected Ca2+ release response to ML‐SA1 was substantially attenuated. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in podocytes. Sphingosine as the AC product of ceramide hydrolysis was found to increase GCaMP3 fluorescence by 3.95 folds in podocytes. Whole‐lysosome patch clamp recording using a Port‐A‐Patch system was performed to study the effects of various AC‐associated sphingolipids on TRPML1 channel activity. After characterization of integrity and purity of isolated lysosomes, we detected ML‐SA1‐induced Ca2+ currents through the isolated lysosome membrane in a dose‐dependent manner. Sphingomyelin, the substrate of ceramide, blocked Ca2+ release induced by ML‐SA1, ceramide had no effects on ML‐SA1‐induced Ca2+ currents, but sphingosine as a product of ceramide hydrolysis via AC remarkably increased ML‐SA1‐induced Ca2+ release by 2.35 folds. Furthermore, interactions of lysosome and autophagosome in podocytes indicated by colocalization of LC3B‐GFP and Lamp‐1‐RFP were inhibited by carmofur. These results suggest that normal function of lysosomal AC and associated production of sphingosine are important for TRPML1 channel activity and Ca2+‐dependent lysosome trafficking.

Support or Funding Information

This study was supported by NIH grants DK54927 to Pin‐Lan Li and HL89563 to Ningjun Li.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Activity Relationship Studies of SK Channel Modulation
Authors: Orfali Razan , Nam Young‐Woo , Viegas Adam , Zhang Miao
Published: June 2021

The small‐ and intermediate‐conductance Ca2+ activated K+ (SK/IK) channels play a fundamental role in the regulation of neurons in the central nervous system. In animal models, SK/IK channel positive allosteric modulators (PAMs) have been shown to be effective in reducing the symptoms of neurological diseases such as ataxia. Ataxia is a lethal neurological disease characterized by lack of balance and incoordination of muscle movements, often as a result of cerebellar or spinocerebellar neurodegeneration. Despite these progresses, current available SK/IK channel PAMs often suffer from low potency and/or poor pharmacokinetic properties. Lack of knowledge about the binding site for the compounds is a major reason that hinders the development of more effective therapeutics targeting SK channels. We previously determined the crystal structures of the PAMs such as 1‐EBIO and NS309 in complex with their binding pocket. We found a strong correlation between the potency of the PAMs and their interaction energy within the binding pocket. These structure‐activity relationship studies of SK/IK channel modulation by PAMs might facilitate drug discovery targeting SK/IK channels.

Support or Funding Information

American Heart Association SDG Grant and National Ataxia Foundation YI‐SCA Grant to M.Z.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Cardiovascular and Renal Effects of Synthetic Nociceptin/Orphanin FQ Receptor Partial Agonists
Authors: Denys Ian B. , Gao Juan , Zaveri Nurulain T. , Kapusta Daniel R.
Published: June 2021

Following discovery of the endogenous opioid‐like peptide, nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP), numerous peptide and non‐peptide NOP receptor ligands have been developed for potential treatment of disease states including Parkinson's, depression, anxiety, and chronic pain. In addition, our laboratory has demonstrated that NOP receptor peptide ligands produce changes in cardiovascular and renal function. However, we have yet to determine whether non‐peptide NOP receptor ligands have similar cardiovascular and renal effects. Therefore, the aim of the present study was to characterize the cardiovascular and renal profile of the non‐peptide NOP receptor compounds AT‐039, AT‐127, AT‐090, and AT‐403 (Astraea Therapeutics).

Methods

Male Sprague‐Dawley rats were surgically implanted with a venous, arterial, and bladder catheter and continuously infused intravenously (i.v.) with isosaline. After stabilization, conscious rats received an i.v. bolus injection of AT‐039, 127, 090, or 403 (30, 100, or 300 nmol/kg) while mean arterial pressure (MAP), heart rate (HR), and urine flow rate (V) were measured for 90 minutes. Urine samples were analyzed for urinary sodium excretion (UNaV) and urinary potassium excretion (UKV).

Results

Following i.v. bolus administration all AT compounds produced a dose‐dependent diuresis with a decrease in MAP, HR, MAP, UNaV, and UKV. Each ligand also produced sedation with the exception of AT‐039. Further studies with AT‐039 demonstrated that i.v. infusion of this ligand produced dose‐dependent effects on water and electrolyte excretion and blood pressure, but did not alter HR or produce sedation.

Conclusions

These findings indicate that i.v. injection of all non‐peptide NOP receptor AT compounds produced a significant diuretic and hypotensive effects. However, sedation and bradycardia were absent with AT‐039 i.v. infusion treatment. Further studies are needed to explore the mechanisms by which these compounds produce their cardiorenal responses.

Support or Funding Information

Funding for this work was supported by Astraea Therapeutics (NZ), R01DA027811 (NZ), R01014026 (NZ), and NIH P30GM106392 (DRK).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impaired Mesenteric Arterial Function of Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rats: Possible Involvement of Small Conductance Calcium‐activated Potassium Channels (SKca)
Authors: Razan Md Rahatullah , Akther Farjana , Wang Der Yu , Shaligram Sonali , Graham James L. , Stanhope Kimber L. , Havel Peter J. , Rahimian Roshanak
Published: June 2021

UC Davis type‐2 diabetes mellitus (UCD‐T2DM) model is a novel and validated model of type‐2 diabetes exhibiting etiology very similar to clinical T2DM seen in humans. This model is characterized by polygenic adult‐onset obesity and diabetes observed in both sexes along with development of insulin resistance, impaired glucose tolerance, and subsequent β‐cell decompensation. We have previously shown that vascular reactivity in mesenteric arteries (MA) of male UCD‐T2DM is altered. Endothelium‐dependent hyperpolarizing factors (EDHF) is an important regulator of vascular tone, especially in small vessels such as MA. The objective of this study was to investigate the contribution of small or intermediate conductance calcium‐activated potassium channels (SKca & IKca) in modulating vascular function of MA in this model. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) in pre‐contracted MA with phenylephrine (PE, 2μM) was measured before and after pretreatment with indomethacin (Indo, 10μm), a cyclooxygenase inhibitor, followed by addition of L‐NAME (200uM), a nitric oxide synthase inhibitor, and then Apamin (1μm), a selective inhibitor of SKca; and TRAM‐34 (1μm), a selective inhibitor of IKca. We demonstrated that the sensitivity to ACh‐induced relaxation was significantly reduced in MA from male UCD‐T2DM rats compared to controls. Addition of L‐NAME reduced Indo‐insensitive vasorelaxation in both control and diabetic animals. However, when compared with that of control, the effect of L‐NAME was much greater in MA of UCD‐T2DM, indicating a reduced contribution of EDHFtype relaxation or an enhanced role of NO in this disease model. The remaining EDV to ACh which is referred to as the L‐NAME/Indo‐insensitive component, or EDHF‐type relaxation was subsequently blocked by inhibiting intermediate (but not small) conductance calcium‐activated potassium channels in MA of diabetic model. In control animals, however, incubation of MA with apamin (a SKca inhibitor) led to a further reduction of EDV. The subsequent addition of TRAM‐34 eventually blunted the EDHF‐type relaxation in this group. These data, for the first time, show that the contribution of EDHF in EDV is reduced in the MA of UCD‐T2DM group, and the down regulation of SKca may be involved. Clearly, further studies will be needed to document the underlying mechanism(s) of altered vascular function in male UCD‐T2DM rats.

Support or Funding Information

Supported by NIH/NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plasminogen Activator Inhibitor‐1 after Acute Myocardial Infarction in Obstructive Sleep Apnea Patients
Authors: Svatikova Anna , Singh Prachi , Kuniyoshi Fatima , Gami Apoor , Somers Virend K.
Published: June 2021

Background

Acute cardiac and vascular events, including myocardial infarction (MI) exhibit a peak onset between 6 AM and 11 AM. Plasminogen activator inhibitor‐1 (PAI‐1) is a main inhibitor of tissue‐plasminogen activator and a key indicator of prothrombotic state. Elevated levels of PAI‐1 have been linked to increased cardiac and vascular risk. Obstructive sleep apnea (OSA) has also been associated with increased risk of MI. Hypoxemia, as well as OSA, increase PAI‐1. We tested the hypothesis that the early morning after awakening is associated with higher PAI‐1 activity in subjects with acute MI who also have OSA.

Methods

We studied 56 subjects after acute myocardial infarction (mean age 60±2 years; BMI 28±1 kg/m2). All subjects underwent complete overnight sleep polysomnography within 2 weeks after MI to assess for presence and severity of OSA. PAI‐1 activity was measured in all 56 subjects, first before sleep (at 9 PM), and then immediately after awakening in the morning (at 6 AM). A third measurement of PAI‐1 was obtained in all subjects at 11 AM.

Results

For the group, PAI‐1 activity almost doubled from 12.5±1.6 U/mL at 9 PM to 22.5±2.5 U/mL after waking at 6 AM and decreased to 14.2±2.4 U/mL by 11 AM (P<0.0001). Fifty seven percent of patients were observed to have OSA, with apnea hypopnea index of >10 events/hour. Sleep apnea did not contribute to further elevation of morning PAI‐1 activity level in acute MI patients (9 PM, 6 AM and 11 AM PAI‐1 activity respectively: no OSA 11.6±2.5 U/mL, 24.1±3.8 U/mL and 15.2±3.5 U/mL; OSA 13.3±2.1 U/mL, 21.1±3.3 U/mL and 13.3±3.3 U/mL; P=0.8 for between group comparison).

Conclusions

Increased PAI‐1 activity in acute MI patients may be an important mechanism underlying the early morning peak in cardiovascular events. The presence of OSA however does not appear to further accentuate the already elevated PAI‐1 activity in acute MI patients. In patients after acute MI, the diurnal variation in PAI‐1, rather than apnea, may drive the impaired fibrinolysis.

Support or Funding Information

‐‐R01 grant HL65176‐‐This study was also supported in part by a gift to the Mayo Foundation by the Philips Respironics Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does Light Pollution Affect the Development of Atherosclerosis?
Authors: Wendroth Robert H. , McGann Eric G. , Howatt Deborah A. , Pendergast Julie S.
Published: June 2021

Exposure to light at night has been steadily increasing since industrialization. Light at night comes from light bulbs, light pollution produced by cities, and more recently from the widespread use of personal electronics (smart phones, tablets, laptops) at night. Previous studies in outbred mice have shown that dim light at night (DLAN) disrupts circadian rhythms and increases body weight, but it is unknown whether DLAN affects the development of cardiovascular disease. In this project, we are testing the hypothesis that DLAN accelerates the development of atherosclerotic lesions in C57BL/6J Apolipoprotein E‐deficient (ApoE−/−) mice. We single‐housed male ApoE−/− mice in light‐tight boxes at 7 weeks of age for 1 week in 12h light:12h dark (12L:12D) and then in DLAN (12h light: 12h dim 5 lux light) for 12 weeks. Control mice were maintained in 12L:12D for 13 weeks. Body weights did not differ between ApoE−/− mice in 12L:12D and DLAN. In 12L:12D, ApoE−/− mice had a robust daily rhythm of eating behavior that peaked during the night. However, after a few days in DLAN, ApoE−/− mice had a low‐amplitude eating behavior rhythm that persisted for the entire experiment. Preliminary data also suggest that ApoE−/− mice in DLAN had increased atherosclerotic lesion area and serum cholesterol levels compared to ApoE−/− mice in 12L:12D. These data suggest that DLAN disrupts the daily rhythm of eating behavior and may also increase atherosclerosis.

Support or Funding Information

This research was supported by the National Institutes of Health awards P20GM103527 and R03DK107851, an American Physiological Society Undergraduate Summer Research Fellowship, and the University of Kentucky. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Cell Pannexin1 Modulates Severity of Ischemic Stroke by Regulating Cerebral Inflammation and Myogenic Tone
Authors: Good Miranda Elizabeth , Eucker Stephanie A. , LI Jun , Bacon Hannah M. , Lang Susan M. , Butcher Joshua T. , Johnson Tyler J. , Zuo Zhiyi , Isakson Brant E.
Published: June 2021

Ischemic stroke is a leading cause of morbidity and mortality in the United States; however, there currently exists only one effective acute pharmacological therapeutic intervention. Purinergic signaling has been shown to regulate vascular function and pathological processes, including inflammation and arterial myogenic reactivity, and plays a role in ischemic stroke outcome. Purinergic signaling requires extracellular purines; however, the mechanism of purine release from cells is unclear. Pannexin1 (Panx1) channels are novel purine release channels expressed throughout the vascular tree that couples regulated purine release with purinergic signaling. Therefore, we examined the role of smooth muscle and endothelial cell Panx1 in cerebral ischemia/reperfusion injury outcomes using conditional cell‐type specific transgenic mice. Deletion of endothelial cell Panx1, but not smooth muscle cell Panx1, significantly reduced cerebral infarct volume 24 hrs post‐reperfusion following a 90‐min ischemic injury. Infiltration of leukocytes into brain tissue and development of cerebral myogenic tone were both significantly reduced when mice lacked endothelial Panx1. Interestingly, Panx1 regulation of myogenic tone was unique to the cerebral circulation, as mesenteric myogenic reactivity and blood pressure were independent of endothelial Panx1. Overall, deletion of endothelial Panx1 mitigated cerebral ischemic injury by reducing inflammation and myogenic tone development, indicating that endothelial Panx1 is a possible novel target for therapeutic intervention of ischemic stroke.

Support or Funding Information

NIH F32 HL131399, NIH P01 HL120840, NCI P30 CA044579‐23

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SUBSTANCE P REGULATES INFLAMMATORY PATHWAYS IN LYMPHATIC MUSCLE
Authors: Kumar Amy , Lee Yang , Muthuchamy Mariappan , Chakraborty Sanjukta
Published: June 2021

Substance P (SP), a neuropeptide that acts through the neurokinin receptors is associated with the sensory innervation of lymphoid tissue and modulates lymphatic contractile function and lymph flow. SP signaling is associated with several inflammatory pathologies. The role of lymphatics in body fluid homeostasis, lipid absorption and inflammation and immunity rely on the contractility of the lymphatic muscle cells (LMCs). We have shown that lymphatic contractile dysfunction is associated with perturbations in inflammatory mechanisms and that SP activates both inflammatory and contractile pathways in lymphatics. However, the specific inflammatory pathways and the resultant cytokines activated by SP in LMCs is unknown. We hypothesized that SP stimulates the pro‐survival AKT and MAPK pathways, and increases pro‐inflammatory cytokines. Cultured rat mesenteric LMCs were treated with SP (1 mM) and total RNA and protein were isolated at different time points (6 hr and 24 hr). Western analysis was carried out to detect the total and phosphorylated forms of AKT, JNK, GSK3b, b‐catenin, FOXO1 and PDCD4. b‐actin was used as control. The relative levels of inflammatory cytokines and chemokines, and specific inflammatory microRNAs (miRNAs) were analyzed by real time PCR. Our results indicate that SP activates the AKT and JNK pathways to significantly increase inflammatory cytokine and chemokine expression, and induces expression of miR 17–5p, miR19b and miR186 in the LMCs. Additionally, SP stimulates expression of genes, involved in lymphatic remodeling. Taken together our data demonstrate that SP activates multiple inflammatory signaling pathways in the LMCs that contribute to an inflammatory response in lymphatics during pathological conditions.

Support or Funding Information

American Heart Association

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Ex Vivo Platform for Studying Angiogenesis in Perfused Microvascular Networks
Authors: Motherwell Jessica , Katakam Prasad , Murfee Walter
Published: June 2021

A challenge in tissue engineering biomimetic models for studying angiogenesis is building the physiological complexity of real microvascular networks. Utilizing an alternative top‐down approach, our laboratory recently introduced the rat mesentery culture model as an ex vivo experimental platform for investigating the multi‐cellular dynamics involved in angiogenesis within an intact microvascular network. However, a current limitation of the model is that microvessels are not perfused. The objective of this study was to demonstrate the feasibility of perfusing microvascular networks during the culture of rat mesentery tissue. Adult Wistar rat mesentery tissues were harvested along with a feeding second order arteriole and venule, and then transferred to a customized tissue chamber for perfusion. The arteriole was cannulated with a glass pipette (40 – 60 μm diameter) and secured in place with 7–0 suture. Injection with blue food dye through the cannulated arteriole identified the lumens of vessels across the hierarchy of intact microvascular networks and confirmed successful perfusion. Tissues with cannulated arterioles were cultured in the perfusion chamber up to three days with either minimum essential media (MEM) or MEM supplemented with 10% serum. Live/dead labeling confirmed that cannulated mesentery tissues remain viable after three days in culture. Additional labeling with BSI‐lectin identified endothelial cells along microvascular networks and confirmed the tissues were undergoing angiogenesis, characterized by increases in vascular density and capillary sprouting. These results demonstrate the ability to perfuse intact microvascular networks ex vivo in cultured mesentery tissues and supports the novelty of the rat mesentery culture model as a biomimetic tool for investigating angiogenesis.

Support or Funding Information

NIH R01AG049821

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.