bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure‐activity relationships of free fatty acid receptors GPR40 and GPR120 agonists based on a docking simulation
Authors: Hara Takafumi , Hirasawa Akira , Takeuchi Masato , Ayukawa Kumiko , Shirai Ryohei , Kimura Ikuo , Suzuki Takayoshi , Miyata Naoki , Tsujimoto Gozoh
Published: June 2021

Free fatty acid receptors (FFARs) are a member of G protein‐coupled receptor (GPCR) family. Among the FFARs, GPR120 and GPR40 whose endogenous ligands are medium‐ and long‐chain fatty acids are thought to play important physiological roles in insulin release. Although they have received increasing attention as attractive drug targets for diabetes, much still remain unclear about their pharmacology and few specific ligand has been reported so far. To develop specific agonists for these receptors, a series of compounds derived from a peroxisome proliferators‐activated receptor γ agonist were synthesized, and their structure‐activity relationships as agonists for these receptors were explored by using homology models of GPR40 and GPR120. We calculated the hydrogen bonding energies between the compounds and these models. The energies correlated well with the agonistic activity of the compounds that were examined as the ERK activity in either cloned GPR120 or GPR40 systems. Furthermore, the compounds with the lowest hydrogen bonding energies for each receptor potently activated glucagon‐like peptide‐1 (GLP‐1) secretion from STC‐1 cells and insulin secretion from MIN6 cells, the cells that endogenously express GPR120 and GPR40, respectively. Taken together, our results showed that a docking simulation using GPR120 and GPR40 homology models might be useful to predict the agonistic activity of compounds.
Targeting degradation pathways of RGS2 using high‐throughput siRNA screening
Authors: Sjogren Benita , Swaney Steven , Neubig Richard
Published: June 2021

Regulator of G protein signaling (RGS) proteins speed GTP hydrolysis by Gi and Gq protein α‐subunits. RGS2 is highly expressed in smooth muscle and selectively regulates Gαq signaling. RGS2 KO mice are hypertensive and show increased responses to angiotensin and α‐adrenergic agonists. RGS2 mutations found in Japanese hypertensive patients reduce protein expression of RGS2 in cells. RGS2 is rapidly degraded, however, the exact mechanism is not known. The aim of this study was to use a high‐throughput siRNA screen to identify genes that regulate protein expression of RGS2. RGS2 protein expression is up‐regulated by the proteasome inhibitors MG‐132 and Lactacystin in HEK‐293T cells and in primary vascular smooth muscle cells. We utilized the DiscoveRx PathHunter™ ProLabel detection assay, a β‐galactosidase complementation assay, that efficiently quantifies levels of proteins tagged with the 4kDa ProLabel tag. This assay was used to screen a druggable genome subset of the Dharmacon siGENOME SMART‐POOL human siRNA library (~3,200 genes) which includes GPCRs, ion channels, kinases, and protein degradation genes. We identified several components of the ubiquitin protein degradation machinery whose knock‐down increased RGS2 protein expression. Increased understanding of RGS2 protein regulation should help identify new RGS2 modulators for cardiovascular disease. (Supported by NIH‐DA023252).
Enhancement of antioxidant capacity by novel dithiolethiones as a consequence of Fyn inhibition
Authors: Koo Ja Hyun , Lee Woo Hyung , Kim Sang Geon
Published: June 2021

Fyn has emerged as a regulator of diverse pathological processes. However, the inhibitor is clinically available. This study investigated the potential of cycloalkane‐fused or other dithiolethiones in increasing antioxidant capacity in association with Fyn inhibition and the molecular basis for this effect. Treatment of HepG2 cells with each agent protected mitochondria from oxidative injury, increasing cell viability; 4,5,6,7‐tetrahydrobenzo‐1,2‐dithiole‐3‐thione (SNU1A) and 5,6‐dihydro‐4H‐cyclopenta‐1,2‐dithiole‐3‐thione were the most effective, whereas 5‐methyl‐1,2‐dithiole‐3‐thione was intermediate. 5‐(Quinolin‐2‐yl)‐1,2‐dithiole‐3‐thione was the least active. SNU1A treatment decreased mitochondrial superoxide production, and restored mitochondrial membrane permeability. Oxidative injury enhanced Fyn phosphorylation, which was diminished by SNU1A. Fyn inhibition contributed to the protection of mitochondria through AMPK, as supported by Fyn's reversal of the effect. Consistently, Fyn overexpression antagonized AMPK activation by SNU1A, strengthening the inhibitory regulation of Fyn on AMPK. SNU1A and congeners had an antioxidant effect and additionally activated Nrf2. In summary, the new dithiolethiones have antioxidant and cytoprotective effect, which may result not only from Fyn inhibition leading to AMPK activation, but from Nrf2 activation.
The cellular and molecular basis for planarian regeneration
Authors: Reddien Peter
Published: June 2021

The ability to regenerate missing body parts is one of the great mysteries of biology. Planarians are flatworms and a classic regeneration model system. The regenerative abilities of planarians are dramatic: following decapitation, a new head is regenerated in a week and an entire animal can be regenerated from a tiny body fragment. A newly developed arsenal of molecular tools have turned planarians into a powerful molecular genetic system for in vivo investigation of the replacement of missing cells. We determined that there exist pluripotent stem cells, called cNeoblasts, that persist into adulthood and provide the cellular basis for planarian regeneration. We have identified dozens of genes required in vivo for normal cNeoblast physiology. How do wound sites specify what is missing? We investigate the instructions that guide cNeoblasts for the regeneration of appropriate missing structures to understand the molecular basis for regeneration.
Endothelium‐Derived Nitroxyl‐Mediated Relaxation Is Resistant To Superoxide Scavenging And Preserved In Diabetic Rat Aorta
Authors: Woodman Owen L , Leo Chen-Huei , Hart Joanne L
Published: June 2021

The aim of this study is to investigate whether acute exposure to oxidative stress or diabetes affects the contribution of HNO to endothelium‐dependent relaxation in the rat aorta. Vascular responses to the endothelium‐dependent relaxant, ACh, the HNO donor, Angeli's salt and the NO donor, DEANONOate, were determined using organ bath techniques. Exposure to the superoxide generator pyrogallol (100 μM) significantly reduced the sensitivity to DEANONOate (pEC50: control, 8.03±0.10 vs. pyrogallol, 7.48±0.09, n=6, p<0.01) but had no effect on responses to Angeli's salt. In the presence of L‐cysteine (a selective HNO scavenger), pyrogallol caused a further inhibition to ACh‐induced NO‐mediated relaxation. In contrast, the addition of pyrogallol to hydroxocobalamin (HXC, a selective NO scavenger) did not cause any further inhibition of ACh‐induced relaxation, indicating that HNO‐mediated relaxation was resistant to superoxide. Similarly, in the diabetic aorta, responses to ACh were not affected when NO was abolished by HXC, indicating a preserved HNO‐mediated relaxation. Conversely, when HNO was inhibited with L‐cysteine, the responses to ACh (pEC50: normal, 7.01±0.04 vs. diabetic, 6.72±0.1, n=7–9, p<0.05), were significantly decreased in diabetic aorta, suggesting that NO was impaired by diabetes. These findings demonstrate that HNO component of relaxation is unaffected by oxidative stress.
Insulin‐mediated Akt/eNOS signaling is defective in ob/ob mouse aorta due to negative regulation by translocated GRK2
Authors: Matsumoto Takayuki , Taguchi Kumiko , Kamata Katsuo , Kobayashi Tsuneo
Published: June 2021

We investigated whether and how, in aortas from ob/ob mice (a model of type 2 diabetes), translocation of G‐protein coupled receptor kinase 2 (GRK2) to membranes regulates insulin‐induced relaxation and NO production. Endothelium‐dependent relaxation was measured in aortic rings. The expression and activities of GRK2, β‐arrestin2, and Akt/eNOS proteins were assayed by Western blotting. In ob/ob aortas [vs. control (Lean) aortas]: (a) insulin‐induced relaxation and NO production were reduced, the ob/ob vs. Lean differences being abolished by Akt‐inhibitor (due to reductions in the Lean group); (b) these deficits were prevented by GRK2‐inhibitor; (c) insulin‐stimulated Akt and eNOS phosphorylations were, or tended to be, decreased (in the membrane fraction, some were markedly decreased); (d) GRK2 protein in membranes was elevated, and was further increased by insulin. In ob/ob aortic membranes, insulin increased GRK2 protein and decreased β‐arrestin2 protein, and the phosphorylation levels of Akt (at Thr308) and eNOS (at Ser1177 and Thr495) under insulin were augmented by GRK2‐inhibitor. This study identifies GRK2 translocation as a key negative regulator of insulin responsiveness in the murine aorta. GRK2 may be an important regulator of the β‐arrestin2/Akt/eNOS signaling pathway, which is implicated in diabetic endothelial dysfunction.
Tie‐2‐Cre‐mediated Inactivation of Lipid phosphate phosphatase (Lpp)‐3 Results in Vascular Defects
Authors: Chatterjee Ishita , Wary Kishore K
Published: June 2021

Rationale

Mouse embryos with null Lpp3 alleles die as early as E6.5days as they fail to form chorio‐allantoic placenta and yolk‐sac blood vessels. Thus, in vivo function of Lpp3 gene during later stages of embryonic development is not known.

Goal

To examine tissue specific function of Lpp3, we generated Lpp3flox/flox mice. To delete Lpp3 in endothelial cell (ECs), we crossed Lpp3flox/flox mice with Tie‐2‐Cre transgenic line.

Results

In contrast to the conventional Lpp3 deletion, Tie‐2‐Cre mediated Lpp3 deletion in ECs did not affect normal vasculogenesis; however, this led to impaired angiogenesis and late embryonic lethal phenotype. Microscopic analyses revealed reduced fibronectin and VE‐cadherin expression, and increased apoptosis in CD31‐positive vascular structures. Transmission electron microscopy confirmed the presence of apoptotic ECs in mutants. Accordingly, Lpp3 null cells showed increase in p53 and p21 levels, with concomitant decrease in cell proliferation as illustrated by reduced 6‐Bromodeoxyuridine incorporation. In vitro, LPP3 knockdown decreased transendothelial electrical resistance, interestingly re‐expression of β‐catenin cDNA into LPP3 depleted ECs partially restored the effect of loss of LPP3.

Conclusion

These results define a critical role of LPP3 in angiogenesis, vascular development and control of apoptotic pathway.

Support: NIH R01HL07935, UIC CCTS UL1RR029879.
Sphingosine Kinase‐1 regulates VEGF‐A induced angiogenesis by mediating the interaction between VEGFR2 and S1P1
Authors: Chavez Alejandra M. , Chava Koteshwara Rao , Mehta Dolly
Published: June 2021

Sphingosine‐1‐phosphate (S1P) and vascular endothelial growth factor A (VEGFA) are critical in angiogenesis, which is characterized by cell responses like migration and proliferation. Sphingosine kinase‐1 (SPHK1) generates S1P from sphingosine and activates sphingosine‐1‐phosphate receptor 1 (S1P1) to maintain endothelial barrier function. Herein, we address the hypothesis that SPHK1 activation of S1P1 is required for mediating angiogenesis by VEGFA. We show that SPHK1 depleted human endothelial cells (ECs) or SPHK−/− ECs fail to migrate and from tube networks. In vivo we also find less blood vessel arborization and lumen formation in Matrigel plugs from SPHK−/− mice. Surprisingly, adding VEGFA to SPHK1 depleted ECs did not rescue the defects. Knock down (KD) of S1P1 prevented VEGFA induced angiogenesis while S1P restored tube formation and migration in SPHK1 KD cells, thus indicating that SPHK1→S1P→S1P1 axis is required for VEGFA induced angiogenesis. Since VEGF receptor 2 (VEGFR2) primarily regulates VEGFA induced angiogenesis we focused on it. We found that SPHK1 KD altered the activation of VEGFR2 downstream effectors and suppressed cell surface VEGFR2 localization. We further show that VEGFR2 pulls down S1P1 in naïve ECs but fails to interact in SPHK1 depleted ECs. Our data identify a novel role of SPHK1 in mediating interaction between VEGFR2 and S1P1 that is required for VEGFA to induce angiogenesis.
Melatonin inhibits NO‐dependent activation of large conductance, calcium‐activated K channels (BKCa) in porcine coronary artery smooth muscle cells
Authors: Shukla Praveen , Sun Chengwen , OˈRourke Stephen
Published: June 2021

We recently demonstrated that melatonin inhibits NO‐induced relaxation of porcine coronary arteries by activating MT2 melatonin‐receptors (JPET 336: 127, 2011). We tested the hypothesis that melatonin inhibits NO/cGMP‐dependent activation of BKCa channels in coronary artery smooth muscle cells. Melatonin (10−7M) inhibited sodium nitroprusside (SNP)‐induced relaxation of isolated coronary arteries but had no effect on relaxation induced by 8‐Br‐cGMP, a stable, cell permeable cGMP analog. ODQ (10−5 M) completely inhibited SNP‐induced relaxation, as well as the increase in intracellular cGMP levels in response to SNP. In patch clamp studies, SNP (10−5 M)‐induced increases in BKCa currents were abolished in the presence of ODQ (10−5 M). Melatonin (10−7M) inhibited both the voltage‐dependent and SNP‐induced increase in BKCa currents, but had no effect on BKCa currents elicited by 8‐Br‐cGMP (10−3M). The inhibitory effect of melatonin on the voltage‐dependent and SNP‐induced increase in BKCa currents was abolished by either the PDE inhibitor, zaprinast (10−5M), or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). Coronary artery relaxation in response to NO involves the cGMP‐dependent activation of BKCa channels. Melatonin impairs NO‐induced relaxation of porcine coronary arteries by inhibiting the cGMP‐dependent activation of BKCa channels in coronary smooth muscle cells. (Supported by NIH HL77204)
Characterization of SERT I172M sensitivity to SSRI metabolites
Authors: Thompson Brent , Denny Ashley D , Ye Wen Rui
Published: June 2021

Altered activity of the serotonin transporter (SERT) has been hypothesized to contribute to the etiology of major depression, anxiety, obsessive‐compulsive disorder, and autism. SERTs are thought to be the primary target for selective serotonin reuptake inhibitor (SSRI)‐class antidepressants, as well as many tricyclic antidepressants (TCA's) used to treat these disorders. Previously, we generated mice expressing a SERT variant (SERT I172M) that displays a dramatic loss of SERT affinity for cocaine, several SSRI's and TCA's, without impacting serotonin (5‐HT) transport (Thompson B.J., et al., 2011). Ex vivo forebrain synaptosomal 5‐HT transport assays with mice carrying the I172M substitution reveal an ~1,000 fold shift in citalopram potency, ~150 fold shift in venlafaxine potency, ~ 60 fold shift in sertraline potency and an ~10 fold shift in fluoxetine potency. Here we demonstrate that citalopram's metabolite, desmethylcitalopram, maintains an ~1000 fold shift in potency for SERT I172M, while the metabolites of venlafaxine, sertraline and fluoxetine display much smaller shifts in potency (~40 fold shift for desvenlafaxine, <2 fold shift for desmethysertraline and <2 fold shift for norfluoxetine). These results highlight the importance of considering the actions of drug metabolites. Ongoing studies are examining the effects chronic SSRIs have on gene expression and behavior.

Grant Funding Source : UTHSCSA Faculty Startup Funds
Chronic fluoxetine treatment is associated with persistent desensitization of the 5HT2A receptor and decreased cocaine‐primed reinstatement in rhesus monkeys
Authors: Sawyer Eileen K , Howell Leonard L
Published: June 2021

Previously, we showed that chronic selective serotonin reuptake inhibitor (SSRI) treatment resulted in a significant suppression of cocaine‐primed reinstatement that persisted after 6 weeks of washout. The therapeutic effects of selective serotonin reuptake inhibitors (SSRIs) for depression are believed to rely on neuroadaptive changes in the serotonin system that emerge during chronic treatment, but these effects have not been evaluated in the context of cocaine use. Therefore, we examined the effects of chronic fluoxetine treatment (10 mg/kg/day, p.o.) in rhesus macaques trained to self administer cocaine and evaluated pre‐and post‐synaptic receptor level and function using positron emission tomography and concurrent in vivo microdialysis and prolactin response. A significant increase in serotonin 2A receptor binding potential was observed, which persisted following a 6‐week washout period. Serotonin transporter binding potential and serotonin release in response to fenfluramine (3.0 mg/kg, i.v.) were not affected, while prolactin release was significantly blunted. Furthermore, cocaine‐induced increases in dopamine and cocaine‐primed reinstatement were also significantly blunted following fluoxetine treatment and washout. Together, these results suggest that chronic fluoxetine treatment is associated with a persistent change in 5HT2A receptor function which may contribute to suppressed cocaine‐primed reinstatement.

This research was supported by NIH grants DA10344 (LLH), DA000517 (LLH), T‐32 DA015040 (support for EKS), and RR00065 (YNPRC).
Tongluo Jiunao Injection, a Chinese herbal fomula protects the focal ischemic brain injury and decreases the expression of Semaphorin 3A and its receptor Neuropilin 1
Authors: Tang Huiling , Zhang Wei , Li Weihong , Tang Yibo , Di Bo , Pan Yanshu , Liu Zhenquan , Li Pengtao
Published: June 2021

Tongluo Jiunao (TLJN) injection has been shown to exhibit protective effects following ischemia injury in vivo and in vitro. We hypothesized that TLJN injection alleviates the injury of neuron and brain blood barrier by inhibiting the expression of Semaphorin 3A (Sema 3A) and its receptor Neuropilin 1 (Nrp‐1) following ischemia stroke. A permanent middle cerebral artery occlusion (PMCAO) surgery was induced in the study on SD rats. Brain edema, infarct volume and spatialtemporal expression of Sema 3A and Nrp‐1 were detected. Triphenyltetrazolium chloride staining results revealed that TLJN injection reduced the infarct volume from 1d to 7d (p<0.01) and edema of brain at 1d (p<0.01) post injury compared to model group. Double‐labeled immunofluorescence showed that Sema 3A and Nrp‐1 positive cells increased in cortex and hippocampus in ipsilateral hemisphere at all the time points after the onset of PMCAO. Enzyme‐linked immunosorbent assay results showed that TLJN decreased Sema 3A. Reverse‐transcription polymerase chain reaction and western blot results showed that TLJN injection decreased the expression of Sema 3A and Nrp‐1 in the ischemic brain tissues of rats. These results suggested that TLJN injection reduced the damage of brain by decreasing the infarction volumn and edema of brain which is related to its inhibitory of Sema 3A and Nrp‐1.
Complement‐derived anaphylatoxin, C5a‐mediated signaling pathway is a novel pharmacological target for IVIG‐regulated humoral immunotherapy in Alzheimer's Disease
Authors: Vempati Prashant , Gong Bing , Zhao Wei , Wang Jun , Devi Lakshmi A , Pasinetti Giulio M
Published: June 2021

Human intravenous immunoglobulin (IVIG) has been found to benefit patients with Alzheimer's disease (AD) and other neurodegenerative diseases. According to a common hypothesis, IVIG contains naturally occurring auto antibodies that block the toxic effects of β‐amyloid (Aβ) and promote brain Aβ clearance. However, with the discrepancy between Aβ pathology in postmortem AD brains and the severity of clinical dementia in the elderly, the beneficial effects of IVIG may extend beyond Aβ clearance. We hypothesized that elevated brain levels of complement C5‐derived C5a anaphylatoxins play a crucial role in the regulation of signaling pathways related to learning and memory functions, which contribute to successful aging, and that human intravenous immunoglobulin may have beneficial effects on cognitive function in age‐related dementia by modulating C5a expression in the brain. Our studies show that the levels of C5a are decreased in C57BL6 mice (24–28 month old), and that reduced brain C5a content is correlated with a decline in cognitive function. Most importantly, four weeks of treatment with IVIG in TG2576 mice significantly increased the level of C5a in the brain and improved synaptic plasticity. Given the high safety of IVIG in clinical practice over the past decade and the lack of effective AD therapy, our study would provide novel mechanisms in AD treatment for the elderly.

Supported by unrestricted grant to GMP
Cognitive and motor effects of endurance related compound AICAR: from muscle to brain
Authors: Kobilo Tali , van Praag Henriette
Published: June 2021

Neurogenesis occurs in the hippocampus, a crucial structure for cognition. Running increases neurogenesis and improves memory. The mechanisms underlying these benefits remain undefined. Transcriptional factors regulating muscle fiber metabolic genes were identified. AMPK is important for mitochondria function and exercise physiology. The agonist AICAR enhanced endurance. The possibility that peripheral triggers, such as skeletal muscle activation as a result of exercise or pharmacological agents, underlies cognitive effects of exercise, has not been explored.

Mice were injected with AICAR for 7 or 14 days. Two weeks later mice were trained in water maze, open field and rotarod. In young mice 7 days of AICAR increased spatial memory, cell survival and percentage of new neurons in the hippocampus. The effects of AICAR were clearly dependent on the duration of administration. In aged mice, 14 days of injections improved motor abilities and cognition, correlated with findings that AMPK activity declines with aging in skeletal muscle. Microarray analysis revealed effects on metabolic processes and muscle composition. Thus, we show that endurance related compounds confer CNS benefits.
Formulation and in vivo evaluation of Floating Microspheres of Misoprostal
Authors: vanitha kondi , Varma Mohan , Alluri Ramesh
Published: June 2021

Prostaglandin (PG) has been reported to be an important protective and acid‐suppressive factor in the gastric mucosa. The objective of the study was to develop and evaluate a stomach specific drug delivery system for controlled release of Misoprostal a PE analogue for gastric ulcer induced NSAIDs. Floating microspheres were prepared by emulsification‐solvent evaporation method using ethylcellulose as a polymer, carbopol as mucoadhesive polymer. Ulcers were induced by the oral administration of absolute ethanol (5 ml/kg) to 24 h fasted Wistar male rats (n =8), weighing 200 g. Sodium bicarbonate solution, misoprostal solution and drug‐loaded microspheres were tested. Formulations were administered orally 1 h before the administration of ethanol. Prior to the oral administration, rats were anesthetized with ethylic ether. After 2 h of ethanol administration, animals were sacrificed; the stomachs were removed, opened along the greater curvature and examined for lesion measurements. Ulcer indexes (UI) were calculated. The in vivo evaluation showed that ulcer index values were 0.61 ± 0.14 for the sodium bicarbonate solution, 0.58 ±0.18 for the misoprostal and 0.11 ± .06 for the misoprostal‐loaded microspheres. The Kruskal‐Wallis test detected statistical differences (p =0.002) between the ulcer indexes. The multiple analyses (Student‐Newman‐Keuls) showed that the misoprostal loaded micro particles presented a gastric ulcer index statistically lower than the sodium bicarbonate solution (p = 0.001) and the misoprostal solution (p = 0.021). The percentages of ulceration inhibition were 31 and 75% after the administration of misoprostal solution and microspheres, respectively. In conclusion, the in vivo evaluation showed that the microspheres presented ulcer index lower than the solutions, showing that misoprostal‐loaded microparticles were efficient in protecting the stomach against ulcer formation.
Pharmacokinetic and pharmacodynamic interactions of indolealkylamine drugs of abuse
Authors: Yu Aiming
Published: June 2021

Indolealkylamine (IAA) drugs are a major class of serotonin analogs that have high impact as substances of abuse. Among them, 5‐methoxy‐N,N‐dimethyltryptamine (5‐MeO‐DMT) was placed into Schedule I controlled substance status in the United States as of January 19, 2011, which is often co‐abused with harmaline. IAA drugs acts mainly on the serotonin neurotransmission system. Overdose of one IAA substrate or concomitant use of two IAA drugs, e.g., harmaline and 5‐MeO‐DMT, causes the excess of 5‐HT or agonists or both, and induces hyperserotonergic effect or Serotonin Toxicity. Monoamine oxidase A (MAO‐A)‐catalyzed deamination is the predominant route for 5‐MeO‐DMT metabolic elimination. In vitro studies using human hepatocytes indicate that concurrent harmaline, an MAO inhibitor (MAOI), blocks 5‐MeO‐DMT substrate depletion. In vivo studies with mouse models show that coadministration of harmaline leads to an increased and prolonged exposure to 5‐MeO‐DMT, as well as a potentiation of 5‐MeO‐DMT‐induced hyperthermia. Interestingly, a more severe hyperthermia is shown in mice treated with 5 mg/kg of harmaline plus 2 mg/kg of 5‐MeO‐DMT than that treated with 10 mg/kg of 5‐MeO‐DMT alone, which is in contrast to the higher exposure to 5‐MeO‐DMT in mice treated with 10 mg/kg of 5‐MeO‐DMT. These findings support that harmaline interacts with 5‐MeO‐DMT not only at the pharmacokinetic level but also at the dynamic level, and concurrent use of MAOI will increase the hazards of 5‐MeO‐DMT intoxication.
Influence of Tamarind Pulp on pharmacodynamics and pharmacokinetics of Gliclazide in Rats/Rabbits
Authors: Kumar K. E. , Kotagiri N. , Katreddi R. R. , Putta S.
Published: June 2021

The study was hypothesized to evaluate the safety of gliclazide therapy in the presence of Tamarind pulp (TP) in rats and rabbits. Studies in rats and rabbits were conducted with oral doses of gliclazide, Tamarind and their combination. Blood samples were analyzed for blood glucose by GOD/POD method and serum gliclazide levels by HPLC method. TP alone reduced blood glucose levels in rats/rabbits and has slightly altered the hypoglycaemic effect of gliclazide when administered together. The serum gliclazide levels were reduced significantly and pharmacokinetic parameters of gliclazide were altered significantly in presence of TP, but did not result in the significant change in blood glucose levels, indicating the reduction in the absorption of TP itself along with absorption of gliclazide. The decrease in the pharmacokinetic parameters like AUC, Cmax, Ka, Cl and increase in the T1/2 indicate the decrease in the absorption and elimination of gliclazide, which might be due to laxative property of TP. The increase in Vd might be due to presence of salt in the TP as preservative. The decrease in the elimination of gliclazide might be due to decreased serum levels, resulted from decreased absorption and increased distribution. The decrease in the serum levels of gliclazide did not result in decreased activity of gliclazide, since TP itself found to produce hypoglycaemia. In conclusion, the combination might be safe with respect to its hypoglycaemic effect, but care should be taken when gliclazide is administered with multiple doses of TP in a clinical situation.
Biotransformation of GS‐1101 (CAL‐101), a Potent and Selective Inhibitor of PI3K delta for the Treatment of Patients with Hematologic Malignancies
Authors: Chen Hao , Evarts Jerry , Webb Heather , Ulrich Roger
Published: June 2021

The objective of this study was to study the biotransformation of GS‐1101 and its metabolites in humans in vitro. Abundant mono‐oxygenated GS‐1101 (GS‐563117 ) was detected by LC/MS/MS in human clinical samples, suggesting oxidation is a major pathway for disposition. Structure elucidation of GS‐563117 revealed the purine moiety to be the principal oxidation site. In vitro approaches were employed to study the metabolism of GS‐1101 to GS‐563117. Oxidation was primarily catalyzed by CYP3A4/5 although xanthine oxidase (XO) also catalyzed the reaction. Biotransformation was prominent in liver microsomes and to a lesser extent in small intestinal, renal, and lung microsomes. In addition to direct excretion, clinical studies indicated that GS‐563117 is also eliminated via glucuronidation. In vitro studies demonstrated that the reaction is primarily catalyzed by UGT1A4. In conclusion, CYP3A4/5‐catalyzed mono‐oxygenation is a primary biotransformation reaction of GS‐1101 although XO also participates in catalysis of the reaction in vitro. In vitro data also indicated that GS‐563117 is further metabolized via UGT1A4‐catalyzed glucuronidation.
Effect of Sulfotransferase Structural Rearrangements in Fulvestrant Sulfation
Authors: Falany Charles Napoleon , Cook Ian T , Kadlubar Susan A , Leyh Thomas S
Published: June 2021

Fulvestant (FUL) sulfation by SULTs 1A1 and 1E1 is influenced by structural rearrangements in the enzymes elicited by PAPS binding. FUL, a chemotherapeutic used to treat estrogen positive breast cancer, is sulfated by both isoforms in vitro; however, FUL sulfation in vivo may be modulated by PAPS levels. PAPS binding to the SULTs results in structural changes that significantly decrease the volume of the substrate binding pocket resulting in differential binding in the presence or absence of PAPS. FUL shows a random Bi‐Bi reaction mechanism with SULT1E1. In contrast, with SULT1A1 FUL displays an apparent ordered reaction mechanism with FUL binding first. FUL docked competently to SULT1E1 in the presence and absence of bound PAP. FUL docked to SULT1A1 only in the absence of PAPS. SULT1E1 bound FUL with a Kd of 0.2 μM in the presence or absence of PAP, whereas FUL bound SULT1A1 with a Kd of 2.4 μM in the absence of PAP and no binding was detectable in the presence of PAP. PAP was an uncompetitive inhibitor of FUL sulfation by SULT1A1 consistent with an ordered reaction mechanism. In summary, PAPS elicited structural changes in SULT1A1 and 1E1 may significantly affect the kinetics and apparent reaction mechanism of FUL sulfation suggesting that cellular PAPS levels may inhibit FUL sulfation in tissues with high SULT1A1 activity. Supported by PHS grants GM38953 to CNF and CA128897 to SAK.
Decrease in Hepatic TNF‐alpha and increase in DNA yield defines hepatoprotection of stem‐bark aqueous extract of Sclerocarya Birrea in ethanol‐induced liver injury in Wistar rats
Authors: Alawa Judith Nkiruka , Ahmadu Sadiya , Kwanashie Helen , Akpulu Peter , Ellah Ezekiel
Published: June 2021

Modulation of the toxicity of tumor necrosis factor‐alpha (TNF‐α) is important in treatment of alcoholic liver damage as it depletes oxygen in hepatocytes leading to damage of biomolecules (DNA) and apoptosis. Sclerocarya birrea (SB) has shown hepatoprotection in ethanol‐induced liver damage. This study investigates whether the stem‐bark aqueous extract of SB will modulate the expression of hepatic TNF‐α thereby preventing oxidative DNA damage in rats exposed to ethanol. Male Wistar rats were exposed to 40% (v/v) ethanol and/or two doses of SB (15 mg/kg or 30 mg/kg) or distilled water for four weeks by gastric gavage. Liver homogenates and tissue were analyzed for TNF‐α, DNA yield and histopathology. Serum was analyzed for antioxidants (superoxide dismutase and reduced glutathione) and liver function enzymes (alanine and aspartate aminotransferases). Ethanol induced hepatotoxicity with marked increase in hepatic TNF‐α and liver function enzymes while antioxidants and DNA yield decreased. Treatment with SB modulated hepatic damage by decreasing levels of TNF‐α, liver function enzymes and increasing antioxidants. Histopathological examination showed that SB attenuated degenerative changes in hepatocytes and liver parenchyma. Results demonstrate that SB modulates TNF‐α expression and induces antioxidant activity thereby preventing oxidative DNA damage and liver injury.

Grant Funding Source: Ahmadu Bello University Research Grant
Effect of low‐dose epicatechin on mitochondrial function and membrane fluidity
Authors: Panneerselvam Mathivadhani , Finley J Cameron , Ali Sameh , Roth David M , Patel Hemal H
Published: June 2021

Epicatechin, a dark chocolate flavonol/antioxidant, is linked to cardiovascular cytoprotection. Myocardial infarct size was decreased in epicatechin‐treated mice compared to controls, and this effect was blocked by a DOR antagonist. Low‐dose epicatechin, with little antioxidant activity, is protective; however, the mechanism of this cytoprotection is unknown. We tested if epicatechin (Epi) mediates cardiac protection via modulation of mitochondrial function and membrane structure. Mitochondria were isolated from all experimental groups [saline control, Epi (1mg/kg), naltrindol (Nal, DOR antagonist, 5mg/kg), Epi+Nal, SNC‐121 (OR agonist, 1mg/kg), and SNC‐121+Nal for 10 days] and assayed for oxygen consumption. Compared to saline controls, Epi and SNC‐121 treated mitochondria showed enhanced functional performance through complex I under state III respiration. This effect was attenuated by DOR antagonism with Nal. Further, we studied the effect of Epi on membrane dynamics. The effect of epicatechin on cardiac myocyte membrane dynamics was examined with electron paramagnetic resonance (EPR) spectroscopy. Epi increased membrane order parameter (decreased membrane fluidity) when compared to vehicle treated myocytes, an effect blocked by Nal. In conclusion, epicatechin alters mitochondrial function and membrane fluidity via DOR to produce cardiac protection.
Regulation of glycogen phosphorylase by the malin‐laforin complex
Authors: Dukhande Vikas V , Bridges Travis M , Gentry Matthew S
Published: June 2021

Autosomal recessive mutations in genes encoding malin or laforin cause Lafora disease (LD), a progressive myoclonic epilepsy. Pathological aberrant glycogen accumulations in brain and various other organs are a hallmark of LD. Our group identified laforin as a novel glycogen phosphatase essential for maintaining glycogen in a soluble form. Malin is a RING‐type E3 ubiquitin ligase. Malin is known to ubiquitinate proteins involved in glycogen metabolism such as laforin, glycogen synthase, protein targeting to glycogen and glycogen debranching enzyme. However, except laforin, expression of these proteins is unchanged in malin knockout mice and thus malin's role in LD pathogenesis is still unclear. Glycogen phosphorylase and glycogen debranching enzyme are vital in catabolism of cellular glycogen. In this study, we aimed to discover substrate/s of malin and its function in LD pathogenesis. We employed immunoprecipitation, western analysis, immunocytochemistry and enzyme activity measurements. Our data uncovered glycogen phosphorylase as a novel substrate of malin. The expression of glycogen phosphorylase was decreased in liver and skeletal muscle tissues from malin knockout mice and glycogen phosphorylase expression was unchanged in tissues from laforin knockout mice. Similarly, overexpression of LD mutations of malin in HEK‐293 cells decreased glycogen phosphorylase expression. In addition, transient overexpression of malin in HEK‐ 293 cells increased the endogenous expression of glycogen phosphorylase. Taken together, our results have identified a novel substrate of malin. Further exploration into the mechanisms of this regulation will unravel insights in glycogen metabolism, physiological functions of malin‐laforin complex and will aid in betterment of LD pharmacotherapy.

Supported by NIH grants R00NS061803, R01NS070899 and University of Kentucky College of Medicine startup funds.
Differential roles for α1‐ versus α2‐ adrenoreceptor activation of mouse mesenteric arterial networks in vivo
Authors: Westcott Erika Boerman , Segal Steven S
Published: June 2021

Isolated segments of mesenteric arteries (MA) are studied widely to define mechanisms of vasomotor control yet little is known of their regulation in vivo. Smooth muscle tone in isolated MA often is induced through adrenoreceptor (AR) activation to study vasodilation; however, the AR subtype distribution in MA networks governing vasomotor tone in vivo is undefined. We tested the hypothesis that α1AR and α2AR differentially effect vasomotor tone in first‐, second‐ and third‐order (1A, 2A and 3A) MA in vivo. In anesthetized female C57BL/6 mice (4 – 6 mo), a MA network was exteriorized and superfused (~5 ml/min; pH 7.4, 37 ºC). Resting diameters (μm) were: 1A, 208 ± 26; 2A, 179 ± 19; 3A, 152 ± 22 and with 10 μM sodium nitroprusside were 219 ± 19, 204 ± 19 & 159 ± 15 (P>0.05; n = 4 – 8, means ± SE). In the presence of propranolol (βAR antagonist, 100 nM), norepinephrine (NE, 1 nM – 10 μM) constricted 1A, 2A and 3A by 51 ± 4, 53 ± 2 & 51 ± 5% (EC50 = 288, 225 and 270 nM). Adding prazosin (α1AR antagonist, 10 nM) reduced NE constrictions to 13 ± 3, 11 ± 1 & 8 ± 2% (P<0.05) while rauwolscine (α2AR antagonist, 100 nM) had relatively little effect. Phenylephrine (α1AR agonist) constricted 1A, 2A & 3A by 51, 55 & 47% (EC50= 210, 180 and 160 nM) while UK 14,304 (α2AR agonist) constricted vessels by 14, 14 & 17% (EC50 = 60, 85 and 193 nM). Thus, vasomotor tone results primarily through activation of α1ARs throughout MA networks in mice. (Support: NIH R01HL086483, R37HL041026)
Exposure to Low Lead Concentration for 7 Days Increases Blood Pressure and Reduces NO Effects in Resistance Arteries
Authors: Vassallo Dalton Valentim , Simões Maylla R , Furieri Lorena B , Broseghini Gilson B , Vescovi Marcos VA , Fiorim Jonaina , Azevedo Bruna F , Salaices Mercedes , Padilha Alessandra Simões , Stefanon Ivanita , Fioresi Mirian
Published: June 2021

We investigated the effects of exposure to lead low concentration for 7 days on blood pressure and mesenteric arteries reactivity, and the role of NO and of the renin‐angiotensin system in the vasoconstrictor response to phenylephrine (PHE). Male Wistar rats (250–300 g) received lead acetate (100 ppm) during 7 days as drinking water or only water. Systolic (SBP), Diastolic blood Pressure (DBP) Heart Rate (HR) were measured. Blood lead concentration and ACE activity were measured. Mesenteric arteries (3rd branch) were mounted in a chamber myograph for measurement of isometric tension and concentration‐response curves to PHE were performed. Lead blood levels attained 5 ± 0.96 μg/dL, below blood lead reference value (60 μg/dL). SBP and DBP increased. The maximum contractile response to PHE did not differ in both groups. Incubation with L‐NAME increased the response to PHE in control but not in lead treated rats suggesting the reduction of endothelial modulation by NO. Enalapril effects and ACE activity did not differ in both groups. Results suggest that low concentration lead exposure after 7 days although increasing blood pressure does not change vascular reactivity but reduced the role of NO in resistance arteries. These results offer further evidence that short time lead exposure at small concentration is hazardous and a risk factor for cardiovascular disease. (CAPES, FAPES/CNPq).
Vitamin C prevents attenuation of flow‐mediated dilation due to acutely altered SR patterns
Authors: Johnson Blair D , Mather Kieren J , Newcomer Sean C , Mickleborough Timothy D , Wallace Janet P
Published: June 2021

Alterations of normal shear rate (SR) patterns at rest impair flow‐mediated dilation (FMD), possibly due to augmented oxidative stress. We tested the hypothesis that the antioxidant vitamin C would prevent the attenuation of brachial artery FMD following a period of altered SR patterns. Twelve lean healthy men (26.3 ± 3.1 years) participated in two trials. One arm was exposed to altered SR profiles using 60 mm Hg of forearm cuff compression for 30 min while the contralateral arm served as the control. Subjects ingested 500 mg capsules of vitamin C or placebo capsules 90 min and 120 min prior to cuff compression in a randomized double‐blind cross‐over design. There were no differences in baseline FMD or antegrade, retrograde, mean or oscillatory SR. For both trials antegrade SR was unchanged in both arms (P > 0.10). Retrograde (P < 0.01) and oscillatory SR (P < 0.01) increased and mean SR (P = 0.01) decreased in the compression arm with no differences between trials (P > 0.23). FMD was unchanged in the control arm in both trials (P > 0.19). FMD decreased following compression + placebo (pre 5.2 ± 1.4% vs. post 3.5 ± 1.4%) (P < 0.01), but remained unchanged following compression + vitamin C (pre 5.3 ± 2.4% vs. post 5.7 ± 2.6%) (P > 0.46). These data demonstrate that acute vitamin C supplementation prevents the attenuation of FMD following altered SR profiles, which indicates that oxidative stress may contribute to the attenuated FMD response.
Microvascular reactivity in subcutaneous and visceral fat tissue in human obesity
Authors: Grizelj Ivana , Cavka Ana , Bian Jing-Tan , Drenjancevic Ines , Phillips Shane A
Published: June 2021

Microvascular dysfunction contributes to cardiovascular disease risk during obesity. Prior data suggest that visceral adipose tissue (VAT) is associated to cardiometabolic risk factors and endothelial dysfunction compared subcutaneous adipose tissue (SAT). The aim of this study was to determine flow induced dilation (FID) in human VAT and SAT resistance arteries. We hypothesized that microvascular FID is impaired in VAT compared to SAT during obesity. Microvessels were obtained from VAT and SAT biopsies in lean (LN; BMI<27) and during bariatric surgery (OB; BMI>48). Isolated vessels were cannulated for vascular reactivity to flow (pressure gradient cmH2O, 20, 40, 60, 100) in the absence and presence of the NOS inhibitor L‐NAME (10−4 M). Dilations to papaverine (10−4M) were determined in each vessel. FID was reduced in VAT (at 60, 59±7%, p<0.05) compared to SAT (at 60, 100±10%). Interestingly, FID was increased in SAT but not VAT from OB compared to LN subjects. There was no change in maximal dilation to papaverine between groups. In addition, there was no effect of L‐NAME on FID in SAT of OB and LN. However, L‐NAME reduced FID in VAT of OB (at 60, 36±8%; p<0.05) vs. baseline. These data suggest that 1) FID is reduced in visceral compared to subcutaneous fat of obese patients and 2) NO mediates FID in microvessels from visceral but not subcutaneous adipose during obesity. NIH (K23HL085614, R01HL095701), UKF (87/11, 88/11).
Downregulation of the α2‐isoform of the Na,K‐pump inhibits EDHF‐like responses in rat mesenteric small arteries
Authors: Aalkjaer Christian , Nielsen Nina Moeller , Matchkov Vladimir
Published: June 2021

Oubain is known to inhibit vascular relaxation mediated by EDHF. Since oubain acts on several isoforms of the Na,K‐pump and can activate a signaling cascade in addition to inhibit ion‐translocation we investigated whether downregulation of the α2‐isoform of the Na,K‐pump affects the EDHF‐like response.

The α2‐isoform of the Na,K‐pump was downregulated with siRNA in rat mesenteric small arteries and the functional effect assessed by isometric myography in arteries constricted with norepinephrine in a concentration providing 80–90% of maximal tone. Downregulation was confirmed at the RNA and protein level

There was no difference in relaxation to acetylcholine between the downregulated and control arteries (maximal relaxation 71±10 and 80±15%, n=7, respectively). After inhibition of NOS and COX less relaxation was seen in downregulated arteries compared to control (36±11 and 78±16%, n=7, respectively, p<0.05). Addition of TRAM34 and apamin fully inhibited this relaxation (6±4 and 4±4%, respectively).

The study demonstrated the importance of the α2‐isoform of the Na,K‐pump for relaxation via EDHF and strongly indicates that the effects of oubain on EDHF is mediated via this isoform of the Na,K‐pump.
Rac1 inactivation and VE‐cadherin junctional disruption contribute to alcohol‐induced endothelial hyperpermeability
Authors: Doggett Travis M , Breslin Jerome W
Published: June 2021

Alcohol intoxication can weaken endothelial barrier integrity. We tested the hypothesis that ethanol inactivates Rac1 and disrupts junctional VE‐cadherin to weaken the endothelial barrier. Transendothelial electrical resistance (TER) of human umbilical vein endothelial cell monolayers served as an index of barrier function, before and after ethanol treatment (5–100 mM). VE‐cadherin localization was determined by immunofluorescence labeling in fixed cells, or longitudinally in live cells expressing GFP‐VE‐cadherin. Rac1‐GTP levels, indicative of activity, were determined by ELISA. We also tested if the Epac1‐specific cAMP analog 8‐CPT‐2′‐O‐Me‐cAMP (8‐CPT), which activates Rac1, could ameliorate ethanol‐induced barrier dysfunction. The results show that ethanol decreases TER in a time‐ and concentration‐dependent manner, concomitant with disruption of VE‐cadherin continuity and formation of small junctional gaps. Ethanol also significantly decreases Rac1‐GTP for up to 60 min. (maximum decrease is 53 ± 5 % of control, p<0.01). Addition of 8‐CPT 5–10 min. after ethanol significantly shortens the recovery time to baseline TER. Our findings suggest that ethanol increases endothelial permeability by inactivating Rac1 and disrupting VE‐cadherin binding between endothelial cells. Supported by NIH R21AA020049, T32AA007577, R01HL098215, and the ABMRF/Foundation for Alcohol Research.
Nanoparticle‐mediated delivery of tetrahydrobiopterin increases nitric oxide synthesis in diabetes
Authors: Meininger Cynthia , Rao Kavitha , Vasir Jaspreet , Dimitrijevic Sanja , Bittenbinder David , Li Xilong , Kelly Katherine , Wu Guoyao , Labhasetwar Vinod
Published: June 2021

A deficiency of tetrahydrobiopterin (BH4), a critical cofactor for endothelial cell (EC) nitric oxide (NO) synthase, may be a common cause of vascular dysfunction in many diseases. Our goal was to augment BH4 levels in ECs in order to increase NO bioavailability and improve vascular function in streptozotocin‐induced diabetic rats. We encapsulated BH4 in biodegradable poly(D,L‐lactide‐co‐glycolide) nanoparticles (NPs) using an emulsion‐solvent evaporation method. Concentrations of BH4 in ECs and vessels were calculated using a reversed phase high‐performance liquid chromatography (HPLC) method after oxidation of samples under acidic and alkaline conditions, while nitrite levels were measured using an HPLC method developed in our lab. NO‐dependent relaxation of vessels was assessed by measuring the response to increasing concentrations of acetylcholine. NPs were taken up by ECs in a time‐and dose‐dependent fashion. BH4 levels in ECs and NO synthesis were increased after uptake of BH4‐loaded NPs. Importantly, BH4‐loaded NPs increased endothelium‐dependent vasodilatory responses of vessels from diabetic rats, demonstrating the ability of NP‐mediated BH4 delivery to restore vascular function. These studies support the feasibility of utilizing biodegradable NPs to deliver therapeutic agents to ECs to attenuate dysfunction associated with vascular disease. (Supported by NIH grant #HL093689).
SKA‐31, an enhancer of SKCa and IKCa channels, increases coronary flow in normotensive rats
Authors: Mishra Ramesh C , Belke Darrell , Wulff Heike , Braun Andrew P
Published: June 2021

Endothelial SKCa and IKCa channels play an important role in the regulation of microvascular tone. We have previously shown that SKA‐31 (naphtho[1,2‐d]thiazole‐2‐ylamine), can inhibit myogenic tone in isolated resistance arteries independent of vasodilatory agonists. We thus hypothesized that acute administration of SKA‐31 may dilate coronary arteries and increase coronary flow. In a Langendorff‐perfused beating rat heart preparation, SKA‐31 dose dependently (0.01–5 μg, single doses) increased total coronary flow (25–30%) in both male (EC50: 0.76 μg) and female (EC50: 0.54 μg) hearts. Increased coronary flow was associated with modest (<20%) increases in left ventricular (LV) pressure and heart rate. Observed changes in flow were more sensitive to SKA‐31 compared to LV pressure and heart rate. The SKA‐31 evoked increases in coronary flow, LV pressure and heart rate were inhibited by a combination of the SKCa blocker apamin and the IKCa blocker TRAM‐34. SKA‐31 evoked vasodilation and associated changes in LV pressure and heart rate were qualitatively comparable to responses evoked by bradykinin (1 μg) and adenosine (10 μg) in the same preparation. Our results demonstrate that SKA‐31 can induce coronary artery dilatation in intact functioning hearts, and further indicate that endothelial SKCa/IKCa channel‐mediated vasorelaxation can be evoked under basal conditions. Supported by CIHR (APB) and NIH (HW).