bluuebunny/crossref_metadata_2025 huggingface dataset:
LC3 Overexpression Perturbs Pancreatic Acinar Cell Homeostasis and Alters Pancreatitis Responses
Authors: Mareninova Olga , Jia Wenzhuo , Elperin Jason , Lotshaw Ethan , Pimienta Michael , Reicher Benjamin , Gukovsky Ilya , Gukovskaya Anna
Published: November 2023

Background & Aims

Despite accumulating evidence for important role of autophagy in pancreatitis, the underlying mechanisms are poorly understood. Conversion of the key autophagy‐related (Atg) protein Atg8/LC3 fromits cytosolic LC3‐I form to the lipidated LC3‐II form is a critical step in autophagosome formation. We used mice (over)expressing LC3 conjugated to green fluorescent protein (GFP‐LC3) to study the role of autophagy in pancreatic acinar cell physiology and pancreatitis.

Methods

Wild type and GFP‐LC3 mice were subjected to fasting (starvation is a strong physiological inducer of autophagy) or used in several dissimilar experimental models of acute pancreatitis, namely those induced by cerulein [an analog of cholecystokin in (CCK)‐8], L‐arginine, choline deficient/ethionine‐supplemented diet, or ethanol diet pluslow‐dose cerulein. We measured parameters of autophagy, acinar cell functions, e.g., amylase secretion, and pancreatitis responses.

Results

LC3 overexpression markedly increased the basal number of autophagic vacuoles in the pancreas; and stimulated starvation‐induced autophagy. In GFP‐LC3 mice subjected to pancreatitis, autophagy was impaired/inefficient, as manifest by accumulation of abnormally large vacuoles and p62‐positive protein aggregates. These effects were quantified by using immunoblot analysis and confocal microscopy of LC3 “puncta”. In addition, the retarded autophagic flux was measured in isolated pancreatic acinar cells by using lysosomalprotease inhibitors. One mechanism underlying these effects could be decreased LC3‐Ito LC3‐II conversion due to feedback down‐regulation of Atg5 and Atg7, which we found in pancreas of GFP‐LC3 mice (both Atg5 and Atg7 mediate the LC3‐I to LC3‐II conversion). In all pancreatitis models studied, LC3 over expression dramatically increased serum amylase levels, a hallmark response of acutepancreatitis. At the same time, pancreatic levels of amylase and trypsinogen were up‐regulated, possibly due to decreased autophagic protein degradation. Another potential underlying mechanism could be an increase in protein synthesis mediated by the mechanistic target of rapamycin (mTOR). Importantly, LC3 over expression worsened pancreatitis severity (e.g., necrosis); but it had no effect on CCK‐induced amylase secretion from acinar cells.

Conclusions

Results indicate a critical role for LC3‐mediated autophagy in regulating exocrine pancreas homeostasis and pancreatitis responses. Although GFP‐LC3 mice are widely used to monitor autophagy, caution should be exercised as LC3 overexpression may perturb both physiologic and pathophysiologic pathways.

Support or Funding Information

NIH/NIAAA R01AA019730, NIH/NIDDK P01DK098108
Induction of AP‐1‐MAPKs Signaling by Copper Oxide Nanoparticles
Authors: Barber Tabatha Lynn , Bowman Linda , Ding Min
Published: November 2023

Occupational exposures to copper dusts or fumes have been reported to be harmful to human health, with possible risk of cancer among copper smelter workers. Copper (II) oxide (CuO) nanoparticles have not, to our knowledge, been extensively examined for potential carcinogenic or genotoxic effects. To investigate the mechanisms of CuO‐induced pathogenesis, the effect of CuO on AP‐1‐MAPKs and ROS generation were investigated. The results indicated CuO caused a 2‐fold increase in AP‐1 activity in JB6 cells. The induction of AP‐1 activity in cultured cell lines was time and dose‐dependent. The signal transduction pathways for AP‐1 activation were also investigated. Western Blot analysis demonstrate that CuO stimulates phosphorylation of p38 MAPK and ERKs. CuO also generated ROS when incubated with the cells as measured by electron spin resonance (ESR). Nano‐sized CuO generated more ROS than the fine sized particles when incubated with the cells. Comet assay suggested that exposure of the cells with CuO resulted in DNA damage. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of CuO‐induced pathogenesis.
Neural Modulation of Liver Function to Regulate Lipid and Glucose Metabolism
Authors: Patel Yogi A , Butera Robert J.
Published: November 2023

Mammalian blood glucose concentrations are maintained within well‐defined biological limits despite considerable fluctuations in the rate at which glucose is obtained from food and utilized by tissues. Of all homeostatic mechanisms, maintenance of blood glucose levels is finely regulated, and one in which the liver and the central nervous system play a prominent role. The liver can add or remove glucose from circulating blood in accordance with the demands of the body, which are transmitted by both hormonal and neural messaging. The hormonal messaging system is well understood, unlike the neural messaging system. Hormonal messaging is mediated by the pancreatic hormones insulin and glucagon, which have opposite effects on blood glucose levels. In contrast, the neural messaging system is controlled via two pathways innervating the liver ‐ one sympathetic and one parasympathetic, both originating from hypothalamic areas. Previous studies have shown that electrical stimulation of these hypothalamic nuclei can lead to rapid changes in blood glucose levels. Additional studies have also shown that electrical stimulation of peripheral nerves innervating the liver can lead to changes in hepatic output, however the limitations associated with neural control of glucose and lipid levels is unclear. Here we take an interdisciplinary approach to characterizing the use of electrical stimulation to control hepatic glucose and lipid metabolism. Using electrical stimulation, we demonstrate and characterize the ability to modulate both sympathetic and parasympathetic activity to the liver, enabling modulation of the glucose clearance rate assessed via oral glucose tolerances tests. Furthermore, we demonstrate how electrical stimulation can modulate lipid metabolism as a function of frequency, amplitude, and location of stimulation.

Support or Funding Information

This work is funded by the GlaxoSmithKline Bioelectronics Initiative.
Activation of dynamin‐related protein 1 contributes to conversion of cardiomyocytes into a proinflammatory phenotype in rat model of sepsis
Authors: Jiang Weipeng , Tao Aibin , Mele Tina , Kao Raymond , Martin Claudio , Rui Tao
Published: November 2023

Dynamin‐related protein 1 (Drp1) is a regulator of mitochondrial fission and cell death pathways. The aim of this study is to determine whether Drp1 plays a role in conversion of cardiomyocytes into a proinflammatory phenotype in sepsis.

Methods

In vivo: rat model of sepsis was induced by intraperitoneally injection of fecal materials and myocardial MPO activity and PMN accumulation were assessed. In vitro: cultured rat cardiomyocytes were challenged with plasma (20%) of septic rats. Myocyte Drp1 activation (Western) and chemokine production (LIX and KC) were determined (RT‐PCR and ELISA).

Results

Six hours after induction of sepsis, PMN infiltration of the myocardial interstitium was evident in the septic rats. This inflammatory response was ameliorated by a Drp1 inhibitor, Mdivi‐1. Challenge of cultured rat cardiomyocytes with septic plasma resulted in Drp1 activation and converted the cardiomyocytes to a proinflammatory phenotype (the myocytes produced and secreted the chemokines, LIX and KC); effects blunted by Mdivi‐1.

Conclusion

The results indicate that Drp1 plays an important role in conversion of cardiomyocytes into a proinflammatory phenotype. The Drp1 could be an effective target for treatment of sepsis. (*Corresponding author)

Support or Funding Information

IRF of Lawson Health Research Institute (IRF 2014‐25); National Natural Science Foundation of China (81370333); Natural Science Foundation of Jiangsu Province, China (BK2015‐1332)
Breaking the TRPV1‐Calcineurin Interaction as a Novel Means for Pain Control
Authors: McAllister Stacy , Zambelli Vanessa , Gross Eric
Published: November 2023

Targeting the transient receptor potential vanilloid 1 (TRPV1) channel for pain control is complex. Previous efforts in drug discovery resulted in failure since TRPV1 agonists are painful and TRPV1 antagonists cause hyperthermia. Calcineurin is an important TRPV1 protein partner since an interaction of TRPV1 with calcineurin favors TRPV1 channel opening. Therefore as an alternative, we hypothesized by breaking the interaction between TRPV1 and calcineurin, we can indirectly modulate TRPV1 to reduce pain sensation.

To test this hypothesis, we used methods including computational biology, peptide synthesis, in vitro, and behavioral pain assays. Based on computational analysis, we discovered a sequence within the TRPV1 C‐terminus that interacts with the calcineurin A subunit. The TRPV1 region forms an alpha coil‐coil, part of the TRPV1 channel inner pore unit, and is evolutionary conserved in mammals. Based on these findings, we synthesized a peptide against this region, linked it to TAT47–57 and called it V1‐cal. In a competitive in vitro calcineurin activity assay, V1‐cal reduced the ability for calcineurin to dephosphorylate a calcineurin substrate as measured by free phosphate release in vitro. Further, we used a carrageenan‐induced inflammatory pain model in male Wistar rats of 10–14 weeks of age. Nociceptive threshold was assessed using the Randall‐Selitto method at baseline and 3 hours after administration of carrageenan. Ten minutes prior to carrageenan, rats were injected intraplantarly with the V1‐cal peptide or saline control. Relative to the control group, rats treated with the V1‐cal peptide had a higher mechanical pain threshold (55±8g vs 36±1g, mean±SEM, n=5/group). Together, these data suggest V1‐cal is an effective therapeutic for pain control by disrupting the TRPV1‐calcineurin interaction.

Support or Funding Information

This work was supported by NIH grant T32 GM089626 to SM.
Calorie Restriction Mimicking by Isoliquiritigenin Attenuates Experimental Diabetic Neuropathy via Induction of Mitochondrial Biogenesis and Autophagy
Authors: Yerra Veera Ganesh , Kalvala Anil Kumar , Kumar Ashutosh
Published: November 2023

Objective

The current study assessed the pharmacological efficacy of SIRT1 activation by a natural compound, Isoliquiritigenin (ILQ) in experimental models of Streptozotocin (STZ) induced diabetic neuropathy (DN) and high glucose induced neurotoxicity in neuro2a cells.

Materials & methods

ILQ was administered at doses of 10 mg/kg and 20 mg/kg to the diabetic animals during 7th& 8thweek after diabetes induction (STZ 55mg/kg, i.p). Animals were subjected to assessment of tactile sensitivity to temperature stimuli and mechanical stimuli. Motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) and Sciatic nerve blood flow (NBF) were determined for functional assessment of neuropathic severity. Sciatic nerves were homogenized for biochemical estimations and western blotting analysis. Nerve sections were processed for tunel assay. For in vitro studies, Neuro2a cells were used and incubated with MEM media containing 25mM glucose to simulate high glucose condition. Cells were treated with ILQ (2.5 & 5 μM) for 24 hrs and then analyzed for reactive oxygen species (ROS) generation, mitochondrial superoxide generation, neurite growth analysis and western blotting analysis.

Results

ILQ administration for 2 weeks ameliorated the diabetes induced hyperalgesia and allodynia as evident from the enhanced tail withdrawal latencies to temperature stimuli and increased paw withdrawal responses to mechanical stimuli in ILQ treated rats. ILQ administration also enhanced MNCV, SNCV and NBF in the diabetic animals. ILQ exposure to neuro2a cells reduced the high glucose induced ROS, superoxide generation and enhanced neurite length. Tunel assay and biochemical analysis showed that ILQ treatment reduced oxidative biomolecular damage by enhancing antioxidant defenses. ILQ treatment also enhanced the reductive NAD pool required for oxidative phosphorylation mediated energy production in the sciatic nerves. Western blotting analysis has shown that ILQ treatment ameliorated the diabetes induced impairment in mitochondrial biogenesis and autophagy through PGC‐1α induction and mTOR inhibition respectively.

Conclusion

ILQ through SIRT1 activation improved mitochondrial biogenesis and autophagic turnover in neuronal cells and imparted mitochondrial protection. Pharmacological modulators of SIRT1 and associated mitochondrial signaling can be developed as potential therapeutic choice for the management of DN.

Support or Funding Information

Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India.
Rapid Antidepressant‐like Effects of the “Uptake‐2” Blocker, Decynium 22 in the Flinders Sensitive Line Rat Model of Depression
Authors: Fraser‐Spears Rheaclare , Owens W. Anthony , Mitchell Nathan C , Overstreet David , Koek Wouter , Daws Lynette C
Published: November 2023

Background

Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed to treat major depression and mood disorders, however, many patients report low to no symptom relief with SSRI treatment. We found that activity of low‐affinity, high‐capacity (uptake 2) transporters for biogenic amines, in particular organic cation transporter 3 (OCT3), may limit therapeutic efficacy of SSRIs. Moreover, in C56BL/6 mice we found that decynium‐22 (D22), which blocks OCT1, OCT2, OCT3 and the plasma membrane monoamine transporter (PMAT), produced antidepressant‐like effects when the serotonin transporter was either pharmacologically or genetically compromised. These studies point to D22 sensitive transporters, putatively OCT3, as a promising target for development of new therapeutics for depression. Here, we expand these studies to examine the antidepressant utility of D22 in a genetically based animal model of depression, the Flinders Sensitive Line (FSL) rat. Importantly, like humans, FSL rats show reduced depressive behaviors after repeated but not acute antidepressant administration. Thus, the FSL rat model can be used to identify novel drugs, or drug combinations, that may have faster acting antidepressant‐like activity and improved therapeutic efficacy.

Methods

Antidepressant‐like behaviors were assessed in adult, male Sprague Dawley (SD) and FSL rats using the forced swim test (FST). Rats were tested 30 min after single saline, fluvoxamine or D22 injections given intraperitoneally. Uptake 2 transporter expression was measured by western blotting techniques and radioligand binding of [3H]D22 in rat hippocampal preparations.

Results

As expected, FSL rats displayed higher baseline levels of immobility in the FST indicating a higher level of depressive behavior compared with SD control rats. Also, as expected, acute injection of the SSRI fluvoxamine (10 mg/kg) reduced immobility time in SD but not FSL rats. Conversely, acute D22 (0.1 mg/kg) administration significantly lowered immobility time in FSL rats, but was not effective in control SD rats. FSL rats also appeared to have elevated expression of hippocampal OCT3 and PMAT total protein measured by western blotting. Measurements of uptake 2 transporter levels via D22 binding are currently in progress.

Conclusions

The reduction in immobility time observed after acute, low dose D22 in FSL rats suggests that OCTs and/or PMAT may be targets for rapidly acting antidepressant drugs, and underscores the utility of animal models for depression. Studies are underway to compare chronic D22 treatment with and without an SSRI in the FSL rat model of depression, with the goal to examine OCT3 and PMAT as targets to develop more efficacious and rapidly acting antidepressants.

Support or Funding Information

NIH MH093320 and NRSA T32DA031115.
Characterization of the discriminative stimulus effects of lorcaserin in rats
Authors: Serafine Katherine M. , France Charles P.
Published: November 2023

Several decades of preclinical research suggest that drugs with agonist effects at serotonin (5‐HT)2C receptors might be useful for treating substance use disorders. Lorcaserin, a drug currently used to treat obesity and under consideration for treating substance use disorders, has agonist properties at 5‐HT2C receptors; however, recent evidence suggests that it might also have agonist effects at other 5‐HT receptor subtypes. The present experiment used drug discrimination, a procedure that is highly pharmacologically selective, to further investigate the mechanism(s) of action of lorcaserin. Male Sprague‐Dawley rats (n=7) discriminated 0.56 mg/kg i.p. lorcaserin from saline while responding under a fixed ratio 5 schedule of food presentation. Lorcaserin (0.178–1.0 mg/kg) dose‐dependently increased lorcaserin‐lever responding. Several direct‐acting 5‐HT receptor agonists and antagonists were examined alone and in combination with lorcaserin. The 5‐HT2C receptor agonist mCPP occasioned greater than 90% lorcaserin‐lever responding in 6/7 rats, as did the 5‐HT2A receptor agonist DOM. The 5‐HT1A receptor agonist 8‐OH‐DPAT occasioned greater than 90% lorcaserin‐lever responding in only 4/7 rats. The 5‐HT2C receptor selective antagonist SB 242084 attenuated lorcaserin‐lever responding in all 7 rats. In contrast, the 5‐HT2A receptor selective antagonist MDL 100907 attenuated lorcaserin‐lever responding in 5/7 rats. In addition to producing directly observable behavior consistent with agonist properties at 5‐HT2C receptors (i.e. yawning), at large doses lorcaserin also induces behavioral effects that are consistent with agonist properties at 5‐HT1A receptors (i.e., forepaw treading). Moreover, when examined in combination with a selective 5‐HT2C receptor antagonist lorcaserin also induced behavior consistent with agonist properties at 5‐HT2A receptors (i.e., head twitching). Taken together, these results suggest that in addition to agonist effects at 5‐HT2C receptors, lorcaserin also has agonist effects at 5‐HT2A and 5‐HT1A receptors. Given that 5‐HT2A receptor agonists can be abused (e.g., LSD) it is crucial to fully understand the behavioral effects of lorcaserin if it is to be considered a potential pharmacotherapy for substance use disorders.

Support or Funding Information

Supported by NIH Grants K05 DA017918 and T32 DA031115.
A Panoramic Measure of Agonist, Modulator, Receptor and Signaling Pathway Selectivity
Authors: Zhang Rumin , Kavana Michael
Published: November 2023

We report a quantitative and panoramic measure of dose‐dependent iso‐efficacy and iso‐occupancy selectivity for G‐protein coupled receptors (GPCRs) based on all measured affinity and efficacy parameters. This novel approach can be used to compare and contrast two agonists, modulators, receptors or signaling pathways, enabling compound rank order as well as on‐ vs. off‐target receptor or signaling bias assessment during drug discovery. In conjunction with endogenous agonist tone and the pharmacokinetic exposure of a candidate drug molecule, this method can be useful to predict efficacy and safety margins.
THE HEPATOPROTECTIVE EFFECTS OF WHOLE PLANT EXTRACTS OF OCIMUM BASILICUM IN EXPERIMENTALLY INDUCED LIVER DAMAGE IN RATS
Authors: Abatan Matthew Oluwole , Asala Margeret Titilayo
Published: November 2023

The liver plays a central role in the metabolism of various endogenous and exogenous substances. This therefore exposes the liver to all kinds of insults produced by many materials including drugs, plants, domestic and industrial chemicals. More than 900 drugs have been implicated with liver injury. There are few medications mentioned to have ameliorating effects on liver damage. However some medicinal plants are reported to have hepatoprotective effects on liver damage.

Ocimum basilicum of the plant family Lamiaceae is widely cultivated as a nourishing food and traditionally used as an antispasmodic, aromatic, digestive, carminative, stomachic, antinephrotoxic and tonic agent. This study was conducted to evaluate the antioxidant and hepatoprotective activity of Ocimum basilicum whole plant extracts using acetaminophen induced liver damage in rats as an experimental procedure using silymarin (100mg/kg) as a reference agent.

Dried homogenized whole plant of O. basilicum was subjected to gradient extraction using chloroform, diethyl ether, ethyl acetate and methanol and the extracts obtained from these tested for hepatoprotective activity by pre‐treating groups of rats with different dosages for 7days before inducing liver damage with acetaminophen at 750mg/kg on the eight day. Liver function test was conducted by determining levels of serum liver enzymes as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, blood urea nitrogen (BUN), total protein and histological studies. The solvent extract with the highest bioactivity was further subject to vacuum liquid chromatography (VLC) and the fractions assayed for their antioxidant properties using DPPH radical scavenging and the ferric reducing antioxidant power assay (FRAP) procedures with ascorbic acid as standard.

Results showed that all the solvent extracts significantly reduced (P<0.05) the levels of the serum enzymes AST, ALT, and ALP with significant reduction (P<0.05) also in BUN, total protein and total bilirubin when compared with silymarin. However the highest reduction was seen with the methanol extract with the experimental rats manifesting only few histopathological changes in their liver compared with those of the rats pre‐treated with the other solvents. The VLC fractions from the methanolic extract produced significant DPPH and FRAP activities when compared with that of ascorbic acid.

The results of the studies suggest that the plant Osimum basilicum may have hepatoprotective with antioxidant activities and supports the folkloric medicinal use of the plant in handling some diseases.
Subacute toxicity of cadmium sulfide quantum dots coated with maltodextrin in vivo
Authors: Gutiérrez‐Sancha Ivonne , Reyes‐Esparza Jorge , Rodríguez‐Fragoso Patricia , Rodriguez‐Fragoso Lourdes
Published: November 2023

In this study, we evaluated the subacute toxicity of cadmium sulfide quantum dots coated with maltodextrin (CdS/MD) in Wistar rats. Quantum dots (QDs) are semiconductor nanocrystals (2–100 nm) with unique optical and electrical properties currently applied in biomedical imaging and electronics industries. One of the more valuable properties of QDs is their fluorescence spectrum, which renders them optimal fluorophores for biomedical imaging. Due to the novel characteristics in fluorescence, QDs represent a family of promising substances to be used in experimental and clinical imaging. Thus far, the toxicity and harmful health effects from exposure to QDs are not recognized, but it is necessary to know the possible toxicity of QDs. For this study, we used male and female Wistar rats were administrated intraperitoneally diary with 200 μg/kg CdS/MD QDs. Control rats received vehicle only. Rats were then sacrificed after 15 days, serum, liver, kidney, heart, spleen, lung, thymus, intestines, stomach and brain samples were collected. Tissue samples were fixed in paraffin for H&E stain and others were fixed and analyzed by fluorescence, with the serum of liver and kidney function tests were performed. The results showed no change in the morphological architecture of the organs analyzed. The observation under fluorescence microscopy samples showed a presence QDs of the organ analyzed. There were no changes in the values of serum liver enzymes compared with the control group, as well as kidney function tests. In conclusion, the CdS‐MD QDs do not produce any damage to organs analyzed in male and female rats Wistar.
Endogenous Substrates for Tumor‐Specific Human Cytochrome P450 CYP2W1
Authors: Zhao Yan , de Montellano Paul Ortiz
Published: November 2023

Human cytochrome P450 enzymes play an important role in the metabolism of steroids, vitamins, eicosanoids, fatty acids and xenobiotics. CYP2W1 is a recently‐discovered human cytochrome P450 enzyme with low or undetectable expression in both adult human liver tissues and extrahepatic tissues, but high expression in human fetal tissues and in many tumor samples, especially colon and adrenal tumors. The distinctive expression pattern of CYP2W1, which features high expression in fetal and tumor tissues, but no expression in normal adult tissues, suggests that the cancer‐specific expression of CYP2W1 might be related to reversion to more primitive, embryonic cell types in which it had specific physiological roles.

In this study we explored the physiological function of CYP2W1 in fetal development and cancer progression by searching for its endogenous substrates. We identified seven endogenous ligands that exhibited binding affinities to CYP2W1: retinoic acid, retinol, retinal, arachidonic acid, farnesol, geranylgeraniol and 17β‐estradiol. CYP2W1 exhibited tight binding affinities for retinoids, which have low nanomolar binding constants, and much poorer binding constants in the micromolar range for the other four ligands. CYP2W1 readily converted all‐trans retinoic acid (atRA) to 4‐hydroxy atRA, all‐trans retinol to 4‐OH all‐trans retinol, and also readily oxidized retinal. The enzyme much less efficiently oxidized arachidonic acid to 8,9‐diHETrE, 11,12‐diHETrE and 14,15‐diHETrE, 17β‐estradiol to 2‐hydroxy‐(17β)‐estradiol, and farnesol to a monohydroxylated product. These findings indicate that CYP2W1 probably plays an important role in localized retinoid metabolism that may be intimately linked to its involvement in tumor development.

Support or Funding Information

This work was supported by NIH grant GM25515.
Chronopharmacology of cisplatin: Role of the circadian rhythm in modulating the cisplatin transporters levels
Authors: Sorensen Daniel N. , Dakup Panshak P. , Gaddameedhi Shobhan
Published: November 2023

Purpose

Cisplatin is one of the most commonly used chemotherapeutic drugs in treating a variety of tumors including cancers of ovaries, testis, lungs, blood and solid tumors of the head and neck. However, the major limitation of cisplatin as a chemotherapeutic drug is due to tumor resistance and nephrotoxicity. The main focus of our project was to study the chronopharmacologic effects that have been demonstrated in human models of cisplatin chemotherapy showing decreased renal toxicity and side effects. We hypothesized a mechanistic, circadian rhythm‐based cause for these outcomes. To further evaluate our hypothesis, we have screened the expression levels of all known cisplatin transporters (both influx and efflux) as well as the cisplatin‐DNA repair activity as a function of the circadian rhythm.

Experimental Design

Studies were done on mouse models of wild‐type and circadian disrupted Per1/2 −/− animals from tissues samples (kidney and liver) taken at different circadian time points and compared via qRT‐PCR analysis for RNA expression along with the same study with human blood samples by the time of day in a 24‐hour period. In case of cisplatin‐DNA repair activity as a function of the circadian rhythm, immuno‐slot blot assays were performed.

Results

Data from kidney tissue RNA samples showed possible clock controlled transporter expression in wild‐type animals as compared to Per1/2 −/− including OCT2, MRP1 (GS‐X) and ATP7A. These mice showed higher expression during the evening hours compared to the morning hours. This was also seen in fresh samples of human blood where the expression levels of these transporters were in opposite phase to mouse tissues. In addition to the circadian variation on expression levels of the above transporters, our findings suggest that cisplatin‐DNA repair function might also be regulated by the time of day in mouse kidney.

Conclusions

These findings indicate a possible mechanism for the chronopharmacology of cisplatin in minimizing the toxicity associated with it and reveal a target for future study of additional mechanistic causes of circadian dosing changes in cisplatin and other medications.

Support or Funding Information

WSU College of Pharmacy Start‐up Funds
Inhibition of Tumor Growth in an Orthotopic Model of Invasive Pancreatic Cancer: A Novel Molecular Pathway Inhibitor
Authors: Cheng Jiongjia , Okolotowicz Karl , Lowy Andrew M. , Mercola Mark , Cashman John
Published: November 2023

In the United States, pancreatic cancer is the fourth leading cause of cancer‐related fatalities and is predicted to increase to number two by 2030. Despite the prevalence, therapeutic options for pancreatic cancer are still limited to surgery and/or combinations of chemotherapy and radiotherapy. Pancreatic ductal adenocarcinoma (PDAC) tumors are the most common and aggressive form of pancreatic cancer and comprise >90% of all pancreatic cancer. The standard systemic chemotherapy for PDAC is gemcitabine with other cytotoxic agents (i.e., paclitaxel), but this treatment only marginally extends a patient's survival rate for < 1 year and has proven to be ineffective. The development of new therapeutic approaches for PDAC is urgently needed. The goal of our work is to characterize a small molecule that suppresses pancreatic cancer progression and develop a drug candidate for pancreatic cancer. Herein, we report a novel small molecule that potently inhibits PDAC tumor growth based on the results from in vitro molecular target and mechanism validation, pharmaceutical characterization and in vivo xenograft studies. Compound 1 very potently induced apoptosis and suppressed PDAC cell proliferation in a variety of PDAC cell types (i.e., LM‐P (a murine PDAC cell line developed by Dr. Andy Lowy at UCSD), MIA PaCa‐2, BxPC‐3) and patient‐derived PDAC cells. In the mechanism of action, lead compound 1 functioned by selectively destabilizing microtubule polymerization as well as activating DNA damage and multiple apoptotic pathways. Moreover, in the presence of lead compound 1, gemcitabine and/or paclitaxel showed enhanced potency to inhibit cellular proliferation of several typical PDAC cells (i.e., LM‐P, MIA PaCa‐2, BxPC‐3). Lead compound 1 is a non‐toxic, pharmaceutically suitable, drug‐like compound with excellent physiochemical properties. Acute administration of compound 1 to mice (1000 mg/kg, 24 h end point) or chronic administration (20 mg/kg/day, 28 days) did not show any abnormal serum clinical chemical values or abnormal pathology. Pharmacokinetic studies showed that compound 1 possessed acceptable bioavailability. An in vivo orthotopic mouse xenograft study using invasive murine PDAC cells (LM‐P) showed that lead compound 1 (20 mg/kg/day, 28 days, i.p.) inhibited pancreatic tumor growth 60% compared to vehicle‐treated mice (at day 29) without any apparent acute or chronic toxicity. Lead compound 1 represents a novel, non‐toxic agent that uses a distinct mechanism of action to treat PDAC. Use of 1 in PDAC therapy is an innovative strategy that will mitigate against unwanted toxic effects otherwise encountered by pancreatic cancer chemotherapeutics.
SAABFAT6‐induced anti‐growth and survival of A549 adenocarcenoma cell is mediated via death and survival pathways and downstream cascades involving regulation of NF‐κB expression
Authors: Kwende Syntia , Oyagbemi Ademola A. , Fagbohun Olusegun A , Omobowale Olutayo T , Adedapo Adeolu A , Yakubu Momoh A
Published: November 2023

We have examined the mechanism of SAABFAT6‐induced antiproliferation and cytotoxic effects on A549 adenocarcenoma cell line. Ethanol extracts of SAABFAT6 (0.5–2 mg/mL) was incubated with A549 cell line and in vitro in the presence or absence of PTK/PKC activator or inhibitor (Ang II, PMA, Tyrphostin, or Calphostin C). Effects of treatments on proliferative/cytotoxic activities or protein expression were evaluated using MTT assay or western blot analysis. SAABFAT6 significantly attenuated A549 cell proliferation at 48 (47%) and 72 (68%) but not at 24 hrs (0.5 mg/mL) and reducing cell growth by ~76% at all‐time points for 1 and 2 mg/mL. For cytotoxic effects, incubation of A549 with SAABFAT6 significantly reduced cell survival by 72%, 86% at 48 and 72 hrs for 1 and 2 mg/mL SAABFAT6 significantly reduction cell survival by 45, 72, and 89% at 24, 48 and 72 hrs, respectively. These effects of SAABFAT6 on A549 growth and survival were potentiated by activator as well as inhibitor of PTK/PKC while significantly attenuating NF‐kB protein expression. We hypothesize that SAABFAT6 is probably acting by interfering with cellular signaling proteins associated with death and survival pathways (PKT/PKC/MAPK) and probably via downstream cascades involving regulation of NF‐κB. Our data may help to identify the most important target molecules for the antiproliferative and cytotoxic actions of SAABFAT6; serve as a novel drug‐supplement in chemotherapeutic regime against adenocarcenoma useful in the prevention and management of lung cancer growth and resistance. Further researches are required to further identify pathway specific mechanisms of SAABFAT6 actions.

Support or Funding Information

Carnegie African Diaspora Fellowship (IIE Grantee ID: 15410197)
Natriuretic Peptide Receptor‐C Agonist Attenuates the Enhanced Expression of Gqα, PLCβ1 Proteins and Vascular Smooth Muscle Cell Hypertrophy From Spontaneously Hypertensive Rats: Role of ROS and ROS‐Mediated Signalling
Authors: Jain Ashish , Anand‐Srivastava Madhu B
Published: November 2023

Hypertension is associated with vascular remodelling due to hyper‐proliferation and hypertrophy of vascular smooth muscle cells (VSMCs). We earlier showed the implication of enhanced expression of Gqα and PLCβ1 proteins in VSMCs from 16‐week‐old spontaneously hypertensive rats (SHR). The present study was undertaken to investigate whether C‐ANP4–23, a natriuretic peptide receptor‐C (NPR‐C) agonist that has been shown to inhibit vasoactive peptide‐induced enhanced protein synthesis in VSMCs, could attenuate VSMC hypertrophy in rat models of cardiac hypertrophy and to explore the underlying mechanisms contributing to this inhibition. For these studies, aortic VSMCs from 16‐week‐old SHR were used. The protein synthesis, a marker of hypertrophy, was determined by (3H) leucine incorporation and the expression of proteins was determined by Western blotting. The protein synthesis was significantly enhanced in VSMC from SHR as compared to WKY and C‐ANP4–23 treatment attenuated the enhanced protein synthesis to WKY control levels. In addition, the enhanced expression of Gqα and PLCβ1 proteins in VSMC's from SHR was attenuated by C‐ANP4–23 treatment. Furthermore, C‐ANP4–23 also attenuated the enhanced levels of superoxide anion (O2−), NADPH oxidase activity, the increased expression of NOX4, p47phox and p22phox, as well as the enhanced phosphorylation of ERK1/2, AKT1/2 and c‐Src. In conclusion, these results indicate that C‐ANP4–23, via the activation of NPR‐C, attenuates VSMC hypertrophy through its ability to decrease the over‐expression of Gqα, PLCβ1 proteins, enhanced oxidative stress and MAPK/AKT signalling pathway. Thus, it can be suggested that C‐ANP4–23, an activator of NPR‐C, may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis

Support or Funding Information

Supported by grants from Canadian Institutes of Health Research and Heart and Stroke Foundation of Canada.
Impaired Vascular Contractility to Uridine Adenosine Tetraphosphate (Up4A) in Angiotensin (Ang) II‐Induced Hypertensive Mice: The Receptor Desensitization?
Authors: Zhou Zhichao , Yadav Vishal R , Sun Changyan , Teng Bunyun , Mustafa S. Jamal
Published: November 2023

We previously showed (Zhou et. al., Vascular Pharmacol. 73:78–85, 2015) that Up4A‐mediated vascular contraction is partly through purinergic P2X1 receptors (P2X1R). Since the plasma level of Up4A is elevated in hypertensives, which implies a crucial role for Up4A‐P2X1R signalling in hypertension. We investigated the vasoactive effect of Up4A in Ang II‐infused mice. Mean arterial blood pressure (MAP) was measured daily in saline or Ang II‐infused mice. Aortas were isolated for isometric tension measurement. Protein expression of Ang II type 1 receptor (AT1) and P2X1R was assessed by Western blot. MAP was elevated by ~50% in 3‐week Ang II‐infused mice indicating hypertension. Protein levels of both AT1 and P2X1R were upregulated in Ang II‐infused aortas. Surprisingly, Up4A (10−9–10−5 M)‐induced concentration‐dependent contraction in saline‐infused aortas was significantly impaired in Ang II group. Both P2X1R antagonist MRS2159 and AT1 antagonist losartan attenuated Up4A‐induced aortic contraction in WT. However, Up4A‐induced aortic contraction with AT1 desensitization by the prior Ang II (100 nM) stimulation in WT was similar to that without the Ang II stimulation, while the second Up4A‐induced aortic contraction was markedly reduced as compared to the first exposure in the same preparation, suggesting a desensitization of P2R. This desensitization was further confirmed by P2X1R desensitizer α, β‐methylene ATP (10 μM). In conclusion, despite the upregulation of AT1 and P2X1R in hypertension, Up4A‐mediated aortic contraction was impaired in Ang II‐infused mice, which was likely through the desensitization of P2X1R but not AT1. This study implies that vascular P2R activity rather than plasma Up4A level may determine the role of Up4A in hypertension.

Support or Funding Information

Supported by HL02339, HL094447 from NIH and MEPSCKL201301 from Sichuan Medical University
Pharmacological evidence that NaHS inhibits the vasopressor responses induced by stimulation of the preganglionic sympathetic outflow in pithed rats
Authors: Sanchez‐Lopez Araceli , De La Cruz Saul Huerta , Gutierrez‐Lara Erika J , Beltran‐Ornelas Jesus H , Centurion David
Published: November 2023

It has been reported that i.v. administration of NaHS, a donor of H2S, elicited dose‐dependent hypotension although the mechanisms are not completely understood. In this regard, several mechanisms could be involved including the inhibition of the vasopressor sympathetic outflow. Thus, this study was designed to determine the potential capability of NaHS to mediate inhibition of the vasopressor responses induced by preganglionic sympathetic stimulation. For this purpose, Wistar rats were anaesthetised, pithed and cannulated for drug administration. In animals pre‐treated with gallamine, the effect of i.v. infusion of NaHS (310 and 560 μg Kg−1 min−1) or its vehicle (phosphate buffer) was determined on the vasopressor responses induced by: (1) sympathetic stimulation (0.03–10 Hz); (2) i.v. bolus injections of exogenous noradrenaline (0.03–3 μg Kg−1); or (3) methoxamine (1–100 μg Kg−1). The vasopressor responses induced by preganglionic sympathetic stimulation were dramatically and dose‐dependently inhibited by i.v. infusion of NaHS (310 and 560 μg Kg−1 min−1), but not by vehicle, particularly at high frequencies. In marked contrast, the vasopressor responses to exogenous noradrenaline or methoxamine were not inhibited by the above doses of NaHS or its vehicle. The above results, taken together, demonstrate that NaHS inhibited the vasopressor responses induced by preganglionic sympathetic outflow by a prejunctional mechanism. This is the first evidence demonstrating this effect by NaHS and may contribute, at least in part, to the hypotension induced by NaHS.

Support or Funding Information

The authors acknowledge to Conacyt for their financial support.
Antibody Targeted Against P2Y12 Receptor Reduces Platelet Activity In vitro and In vivo
Authors: Hensch Nicole R , Karim Zubair A , Khasawneh Fadi T
Published: November 2023

Platelet hyperactivity is known to give rise to thrombotic disorders, and many antiplatelet therapies are designed to reduce the risk of thrombogenesis. ADP is an important platelet activator and activates platelets by binding to the P2Y1 receptor or P2Y12 receptor, both of which are G‐coupled protein receptors located on the platelets’ surface. To this end, although the involvement of the P2Y12 platelet receptor in thrombosis is well characterized, there are currently no parenteral drugs targeting these receptors in clinical use to complement existing antiplatelet therapies. In this study, we designed a parenteral agent, namely an antibody targeted against the P2Y12 receptor ligand biding domain (abbreviated as P2Y12Ab). In vitro studies revealed that the addition of P2Y12Ab could effectively inhibit aggregation induced by ADP. P2Y12Ab was also found to inhibit aggregation activated by U46619 and thrombin. Using FACS analysis, reduced P selectin and Annexin V exposure and reduced integrin activation was also evident in the presence of P2Y12Ab. As for its in vivo activity, the P2Y12Ab also demonstrated inhibitory effects in a FeCl3‐induced thrombosis model, producing a prolonged occlusion time; however, a lengthened bleeding time was also observed. Overall, our findings suggest that P2Y12Ab functionally blocks platelet activity, further indicating that the P2Y12 platelet receptor plays a central role in thrombogenesis. These studies also validate the use of P2Y12Ab as a purposed parenteral antiplatelet agent to be implemented in conjunction with or as an alternative to current antiplatelet strategies.

Support or Funding Information

This research was supported by funds provided by College of Pharmacy, Western University of Health Sciences (to F.T.K).
Hyperpolarization of Mouse Pulmonary Artery Smooth Muscle and Endothelium by Calcitonin Gene Related Peptide
Authors: Norton Charles , Segal Steven
Published: November 2023

The goal of these experiments was to develop mouse pulmonary arteries (PAs) as a viable model for investigating the electrophysiological actions of the sensory neurotransmitter calcitonin gene related peptide (CGRP) on membrane potential (Vm) and electrical signal transmission in PA smooth muscle cells (SMCs) and endothelial cells (ECs). Intact PAs were isolated from anesthetized male C57BL/6J mice (3 months old). Immunostaining confirmed that PAs were sparsely innervated by sensory nerves containing CGRP. For intracellular recording, intact PAs (diameter, ~150 μm) were cannulated, pressurized to 16 cm H2O and studied at 37°C. Alternatively, SMCs were gently dissociated to obtain intact endothelial tubes studied at 32°C. We tested the hypothesis that CGRP would hyperpolarize SMCs and ECs in a concentration‐dependent manner. During continuous recording of Vm, increasing [CGRP] from 1 nM to 1 μM hyperpolarized SMCs of intact PAs from −34±2 mV (rest) to −52±3 mV and EC of tubes from −37±4 mV (rest) to −58±4 mV (n ≥ 4 per group); respective EC50 values were 6.2 and 6.8 nM. With dual simultaneous intracellular recording in endothelial tubes, current injection (±0.5 nA) into one EC evoked corresponding Vm responses (±5 mV) in a remote EC (separation distance, 500 μm), confirming electrical signal transmission along PA endothelium. These preliminary studies confirm that CGRP effectively hyperpolarizes SMCs and ECs in mouse PA while validating the utility of this model for investigating the nature of electrical signaling in response to sensory neurotransmission along the PA wall.

Support or Funding Information

NIH R37‐HL041026
Aging Impairs Spontaneous Ca2+ Signaling and Regulation by CGRP in Smooth Muscle Cells of Mouse Mesenteric Arteries
Authors: Boerman Erika M , Segal Steven S
Published: November 2023

Advancing age is associated with impaired vasomotor function in resistance arteries. Thus, diminished potency of calcitonin gene‐related peptide (CGRP) coupled with loss of sensory nerve inhibition of sympathetic vasoconstriction contribute to impaired vasodilation in mouse mesenteric arteries (MAs; PMC3607869). In light of multiple roles for Ca2+ signaling in governing vasomotor function, we tested the hypothesis that aging impairs both spontaneous and CGRP‐mediated Ca2+ signals in smooth muscle cells (SMCs). MAs (n = 14–18) from Young (~4 months) and Old (~24 months) C57BL/6 mice were isolated, pressurized to 100 cm H2O and studied at 37°C following loading of SMCs with Fluo‐4 (10 μM, 1h, 37 °C ). With ~15 SMCs per field, under control conditions Young MAs had 9 ± 1 sites producing spontaneous local Ca2+ events versus 4 ± 1 sites in Old MAs (P<0.05) while the number of SMCs exhibiting spontaneous Ca2+ waves (7 ± 1) was not different between age groups. Age‐defined EC50 concentrations of norepinephrine (NE; 167 and 600 nM) reduced local Ca2+ event sites in Young and Old MAs (to 3 ± 1 and 1 ± 1, respectively; P<0.05) and increased the number of SMCs with Ca2+ waves in both age groups (to 11 ± 1 and 14 ± 1). In Young but not Old MAs, addition of CGRP (100 nM) restored local Ca2+ event sites and Ca2+ waves to control values. Across age groups the frequency (0.6 ± 0.01 Hz), amplitude (1.3 ± 0.01 F/Fo), rise time (73 ± 8 ms), and duration (FDHM, 164 ± 9 ms) of local Ca2+ events did not differ with age, NE or CGRP treatments. We suggest that the impaired ability of CGRP to restore local Ca2+ signaling (e.g., as associated with BK channel activation) in SMCs of Old MAs during adrenoreceptor activation contributes to age‐related vascular dysfunction underlying impaired vasodilation.

Support or Funding Information

NIH F32HL118836 & R37HL041026
Endothelial Conduction of Rapid Onset Vasodilation (ROV) in Mouse Skeletal Muscle
Authors: Sinkler Shenghua Y , Segal Steven S
Published: November 2023

In response to skeletal muscle contractions, ascending vasodilation (AVD) of feed arteries (FA) increases muscle blood flow and reflects signals initiated downstream from arterioles embedded within active muscle fibers. Muscular activities range from moderate and rhythmic to intense and brief contractions. We tested the hypothesis that the mechanism of AVD differs with the nature of skeletal muscle contraction. Using intravital microscopy, we studied the resistance microvasculature in the gluteus maximus muscle (GM) of anesthetized male C57BL/6J mice (age: 3–4 months; n=6). Under control conditions, a single maximal tetanic contraction (100Hz, 500ms) evoked ROV in primary arterioles (1A) and FA, increasing internal diameter from 30±2μm to 47±2μm in 1A and from 42±2μm to 54±3μm in FA (onset < 1s, peak within 4s). In contrast, during moderate rhythmic contractions at 4Hz, 1A and FA dilations required 10–15s to begin and attained steady‐state diameters of 45±2μm (1A) and 53±4μm (FA) by ~30s. Local stimulation of 1A with acetylcholine (ACh) microiontophoresis (2μm tip, 500ms) increased diameter locally (to 47±3μm) and evoked FA dilation upstream (to 56±3μm). Light‐Dye Treatment (LDT) to disrupt the endothelium midway between 1A and FA eliminated ROV in FA. LDT also abolished FA dilation to ACh microiontophoresis onto 1A downstream while 1A dilations to rhythmic contractions and to ACh remained intact. Remarkably, FA continued to dilate in response to rhythmic contractions following LDT until nitric oxide (NO) synthesis was inhibited (L‐NAME; 10−4M). We conclude that AVD of FA in response to brief intense skeletal muscle contraction (i.e., ascending ROV) reflects intercellular conduction along the endothelium upon initiation in downstream arterioles. In contrast, AVD during rhythmic contractions is mediated by NO release during sustained functional hyperemia.

Support or Funding Information

AHA 15PRE22840000; NIH R37‐HL041026